Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism

Gemcitabine (GEM, 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl--cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.     

Adult Stromal Cells Derived from Human Adipose Tissue Provoke Pancreatic Cancer Cell Death both In Vitro and In Vivo

Background

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.

Principal Findings

Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.

Conclusion

These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Inhibition of Akt potentiates 2-DG-induced apoptosis via downregulation of UPR in acute lymphoblastic leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.     

Rasfonin,a novel 2-pyrone derivative,induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [³H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC₅₀=5.5 μM) than BxPC-3 cells (IC₅₀=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity.     

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

The Glycolytic Inhibitor 2-Deoxyglucose Activates Multiple Prosurvival Pathways through IGF1R

Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a prosurvival oncoprotein, AKT, through PI3K. In this study, we discovered that 2-DG treatments disrupted the binding between insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP3) so that the free form of IGF-1 could be released from the IGF-1·IGFBP3 complex to activate IGF-1 receptor (IGF1R) signaling. Because IGF1R signaling is involved, PI3K/AKT constitutes only one of the prosurvival pathways that are activated by 2-DG treatment; we validated that MEK-ERK signaling was also induced in an IGF1R-dependent manner in some cancer cell lines. Furthermore, our phospho-specific antibody microarray analysis indicated that 2-DG up-regulated the phosphorylation of 64 sites within various signaling pathways in H460 cells. Chemical inhibition of IGF1R reduced 57 of these up-regulations. These data suggest that 2-DG-induced activation of many survival pathways can be jointly attenuated through IGF1R inhibition. Our in vitro analysis demonstrated that treatment with a combination of subtoxic doses of 2-DG and the IGF1R inhibitor II reduced cancer cell proliferation 90% and promoted significant apoptosis.Cancer cells display high rates of aerobic glycolysis in comparison with their nontransformed counterparts (i.e. the Warburg effect (1)). Whether increased aerobic glycolysis drives tumor formation or merely represents a byproduct of oncogenic transformation has been a subject of controversy. Two recent studies demonstrated that the Warburg effect can be reversed in some cancer cells by either the depletion of lactate dehydrogenenase A or switching pyruvate kinase expression from M2 to M1 isoform (2, 3). Interestingly, the reversal of the Warburg effect correlates with a reduction in the ability of the isogenic cancer cells to form tumors in nude mouse xenografts. Viewed in combination, these observations appeared to indicate that tumor cells preferentially use glucose for purposes other than oxidative phosphorylation and that aerobic glycolysis is a valid target for cancer therapeutics.Targeting glycolysis for cancer treatment has been explored previously as a therapeutic approach (4, 5). Of all the glycolysis inhibitors that were evaluated, 2-deoxyglucose (2-DG)³ is the one that has been best characterized in animal model studies and human clinical trials (6–8). It is converted by hexokinase to phosphorylated 2-DG, which becomes trapped inside the cell and inhibits hexokinase (9). As a direct consequence of 2-DG treatment, intracellular ATP is depleted (10, 11), which ultimately suppresses cell proliferation in vitro (12, 13). Nonetheless, the implementation of 2-DG as an anticancer agent in vivo has been a disappointment. Whereas 2-DG suppresses cell growth in vitro, studies using xenografts indicate that 2-DG treatment, when provided as a single agent, does not inhibit tumor growth (6).Because 2-DG is a small molecule, we suspected that it activates other signaling pathways and decided to evaluate its off-target effects. Our initial findings indicated that 2-DG activates AKT function through phosphatidylinositol 3-kinase (PI3K) and is independent of glycolysis or mTOR inhibition. Thus, the inhibitory effect on growth produced by 2-DG-mediated glycolysis inhibition may be partial offset by the fact there is also 2-DG-induced AKT activation (14). In the current study, we used a phospho-specific antibody array to identify IGF1R as the upstream receptor tyrosine kinase that is responsible for the activation of AKT signaling. Using recombinant IGF-1 and IGFBP3 proteins, we discovered that the inhibition of IGF-1 by IGFBP3 is disrupted in the presence of 2-DG. As 2-DG treatment activates IGF-1 signaling, we evaluated other prosurvival signaling pathways such as ERK signaling, which was also activated by 2-DG treatment in some cancer cell lines. Lastly, we tested to see whether an inhibitor of IGF1R would interfere with the prosurvival pathways and increase apoptosis if given in combination with 2-DG.     

Britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by targeting PD-L1 via abrogation of the crosstalk between Myc and HIF-1α in cancer

BackgroundProgrammed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects.PurposeIn this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells.MethodsIn vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model.ResultsBritannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model.ConclusionBritannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.     

Berberine combined with 2-deoxy-d-glucose synergistically enhances cancer cell proliferation inhibition via energy depletion and unfolded protein response disruption

Background

Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy-d-glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth.

Methods

The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression.

Results

BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds.

Conclusions

Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR.

General significance

Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process.     

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

AKT Inhibitors Promote Cell Death in Cervical Cancer through Disruption of mTOR Signaling and Glucose Uptake

Background

PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability.

Experimental Design

Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with ¹⁸F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay.

Results

Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment.

Conclusions

The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.     

Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene

Background

Cancer cells possess unique metabolic phenotypes that are determined by their underlying oncogenic pathways. Activation of the PI3K/Akt/mTOR signaling cascade promotes glycolysis and leads to glucose-dependence in tumors. In particular, cells with constitutive mTORC1 activity secondary to the loss of TSC1/TSC2 function are prone to undergo apoptosis upon glucose withdrawal in vitro, but this concept has not been tested in vivo. This study examines the effects of restricting glucose metabolism by pharmacologic and dietary means in a tuberous sclerosis complex (TSC) tumor xenograft model.

Results

Tumor-bearing mice were randomly assigned to receive unrestricted carbohydrate-free ("Carb-free") or Western-style diet in the absence or presence of 2-deoxyglucose (2-DG) in one of four treatment groups. After 14 weeks, tumor sizes were significantly different among the four treatment groups with those receiving 2-DG having the smallest tumors. Unexpectedly, the "Carb-free" diet was associated with the largest tumors but they remained responsive to 2-DG. PET imaging showed significant treatment-related changes in tumor ¹⁸fluorodeoxyglucose-uptake but the standard uptake values did not correlate with tumor size. Alternative energy substrates such as ketone bodies and monounsaturated oleic acid supported the growth of the Tsc2-/- cells in vitro, whereas saturated palmitic acid was toxic. Correspondingly, tumors in the high-fat, "Carb-free" group showed greater necrosis and liquefaction that contributed to their larger sizes. In contrast, 2-DG treatment significantly reduced tumor cell proliferation, increased metabolic stress (i.e., ketonemia) and AMPK activity, whereas rapamycin primarily reduced cell size.

Conclusions

Our data support the concept of glycolytic inhibition as a therapeutic approach in TSC whereas dietary withdrawal of carbohydrates was not effective.     

Design and synthesis of functionalized coumarins as potential anti-austerity agents that eliminates cancer cells' tolerance to nutrition starvation

Human pancreatic tumor cells have inherent ability to tolerate nutrition starvation which enables them to survive in the hypovascular tumor microenvironment. Discovery of agents that selectively inhibit the cancer cells’ tolerance to nutrition starvation leading to cancer cell death is a new anti-austerity approach in anti-cancer drug discovery. A series of coumarins derivatives were synthesized and evaluated for their anti-austerity activity against PANC-1 human pancreatic cancer cell line. The compound 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (3-phenylpropyl)amide (2c) showed highly potent selective cytotoxicity against PANC-1 cells under nutrient-deprived conditions, with a PC₅₀ value of 0.44 μM, without exhibiting toxicity in normal, nutrient-rich medium. Compound 2c caused dramatic alterations in PANC-1 cell morphology, leading to cell death. The compound 2c was found to inhibit PANC-1 cell migration and colony formation in a concentration-dependent manner. The compound 2c is a lead structure for the anti-austerity drug development against pancreatic cancer.     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.