Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

Differential effects of N-TiO2 nanoparticle and its photo-activated form on autophagy and necroptosis in human melanoma A375 cells

The manipulation of autophagy provides a new opportunity for highly effective anticancer therapies. Recently, we showed that photodynamic therapy (PDT) with nitrogen-doped titanium dioxide (N-TiO₂) nanoparticles (NPs) could promote the reactive oxygen species (ROS)-dependent autophagy in leukemia cells. However, the differential autophagic effects of N-TiO₂ NPs in the dark and light conditions and the potential of N-TiO_2-based PDT for the treatment of melanoma cells remain unknown. Here we show that depending on the visible-light condition, the autophagic response of human melanoma A375 cells to N-TiO₂ NPs switches between two different statuses (ie., flux or blockade) with the opposite outcomes (ie., survival or death). Mechanistically, low doses of N-TiO₂ NPs (1-100 µg/ml) stimulate a nontoxic autophagy flux response in A375 cells, whereas their photo-activation leads to the impairment of the autophagosome-lysosome fusion, the blockade of autophagy flux and consequently the induction of RIPK1-mediated necroptosis via ROS production. These results confirm that photo-controllable autophagic effects of N-TiO₂ NPs can be utilized for the treatment of cancer, particularly melanoma.     

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

We have examined H₂O₂ production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1β (IL-1β) stimulation and B16 melanoma cell adhesion. Production of H₂O₂ was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2′, 7′-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1β treatment there was a 6-fold increase in endothelial cells producing H₂O₂ (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H₂O₂ content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H₂O₂ production intensity was largely heterogeneous in IL-1β-activated HSE. Enhancement of H₂O₂ production by IL-β-treated HSE started at the 4th h and peaked 2–3 h later. The addition of increasing concentrations of IL-1β to HSE for 4 h caused the progressive activation of H₂O₂ production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1Ra) 10 min before IL-1β treatment abrogated IL-1β-mediated enhancement of H₂O₂. From the 2nd h of B16 melanoma adhesion to HSE there was a significant (P < 0.05) enhancement of H₂O₂ content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H₂O₂ production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 μg/ml anti-mouse IL-1β neutralizing antibody reduced the melanoma cell-induced HSE production of H₂O₂ by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H₂O₂ compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1β (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H₂O₂. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells. © 1996 Wiley-Liss, Inc.     

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of ³HOH from D-[5-³H]glucose and ¹⁴C-labelled CO₂, acidic metabolites and amino acids from D-[U-¹⁴C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-¹⁴C]glucose oxidation and D-[5-³H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.     

T‐type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca²⁺) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G₁‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca²⁺ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.     

Prognostic significance of expression of antigens on melanoma cells

Summary The expression of antigens on 33 human melanoma cells obtained directly from surgically excised tumours was investigated by means of antibody-dependent cell-mediated cytotoxic assays. Antisera used in the study were two antisera from human melanoma patients against different tumour-associated antigens on melanoma cells and antisera against carcinoembryonic antigen (CEA) and ₂ microglobulin (₂M). Considerable heterogeneity was observed in the expression of both melanoma-associated and non-melanoma antigens on melanoma cells from 33 different patients.Patients whose tumours were reactive with the melanoma-associated antiserum (CHI) had a significantly longer remission period to stage 2 melanoma. The period to development of stage 3 melanoma also appeared longer, but this was not statistically significant with the number of patients available for study. The expression of CEA, ₂M, and the tumour-associated antigen TIN was not significantly related to the recurrence-free interval. There appeared to be a reciprocal expression of the two melanoma-associated antigens, and patients with tumours expressing CHI but not TIN had a significantly longer recurrence-free interval than patients whose tumours had the opposite antigenic pattern.In the limited number of patients available for study the expression of the antigen CHI did not appear related to thickness of the primary tumour or to immune response of the patients to melanoma cells in leucocyte-dependent antibody and natural killer cell assays. Although the nature of the association between expression of this antigen and longer remission-free period is unknown these results suggest that the expression of certain melanoma antigens on the cell surface may be an important additional variable which has prognostic and therapeutic importance.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

In vitro modulation of human and murine melanoma growth by prostanoid analogues

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16, 16-dimethyl PGA₁, 16,16-dimethyl-PGA₂, 16,16-dimethyl-18-oxa-PGA₂ and trans-δ-2-15-α acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD₂ (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA₁ and PGE₁ were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD₂ was the most active prostaglandin, 2–3 times more potent than PGA₁; PGE₁ was a very weak inhibitor.     

Role of Melanin as a Scavenger of Active Oxygen Species

The protective role of melanin, either synthetic or derived from a metastatic lung melanoma nodule, was studied in terms of its ability to interact with active oxygen species (O₂, H₂O₂, RO, ROO, etc.). Both melanins showed the ability to react with O₂. The superoxide dismutase-like activity corresponds to 21 and 10 U/mg for synthetic and tumor melanin, respectively. The latter value accounts for about 8% of the superoxide dismutase activity of cultured melanoma cells. Neither type of melanin showed catalase-like or glutathione peroxidase-like activity. Both types of melanin reacted with RO and ROO radicals as determined by inhibition of the lipid peroxidation reaction of rat liver homogenates. The spontaneous lipid peroxidation of rat liver homogenate was inhibited up to 90% and 80% by synthetic and tumor melanin with half-maximal effects at 2.5 and 5.5 μg melanin/ml, respectively. The 2,2-azobis-(2 amidino propane) (AAPH)-initiated lipid peroxidation of rat liver homogenate was inhibited up to 3% and 20% by synthetic and tumor melanin, with half maximal effect at 120 and 500 μg melanin/ml, respectively. Both types of melanin were able to protect the in vitro inactivation of glucose oxidase, which occurs in the presence of AAPH-generated radicals.     

Enhancement of cytotoxic and proliferative responses of lymphocytes from melanoma patients by incubation with monoclonal antibodies against ganglioside GD3

Summary Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD₃ may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD₃ may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD₃. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4⁺ and T8⁺ clones. The potentiation of these responses was not specific for M.Abs to GD₃ but was also seen with M.Abs to GD₂ and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD₃ during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD₃ but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo     

Generation of human-melanoma-specific T lymphocyte clones defining novel cytolytic targets with panels of newly established melanoma cell lines

Melanoma is a cancer where the immune system is believed to play an important role in the control of malignant cell growth. To study the variability of the immune response in melanoma patients, we derived melanoma cell lines from several HLA-A2⁺ and HLA-A2^– patients. The melanoma cell lines studied were designated FM3, FM6, FM9, FM28, FM37, FM45, FM55_P, FM55_M1 and FM55_M2 and were established from eight metastatic tumors as well as from one primary tumor from a total of seven different patients. On the basis of the ability of tumor cells to induce specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes (PBL) in mixed lymphocyte/tumor culture with HLA-A2⁺ melanoma cells, the FM3 cell line was characterized as highly immunogenic. To investigate the expression of different melanoma-associated antigens recognized by CTL on different melanoma cell lines, we selected the cell line FM3 for restimulation and further T cell cloning experiments. The lytic activity of CTL clones with good proliferative activity was examined using a panel of HLA-A2⁺ and HLA-A2^– melanoma cell lines. None of the tested HLA-A2^– melanoma cell lines were susceptible to lysis by the CTL clones, whereas allogeneic HLA-A2⁺ melanoma cell lines were lysed only by a few CTL clones. On the basis of their reactivity with different melanoma cell lines, it was possible to divide the present CTL clones into at least four groups suggesting the recognition of at least four different antigens. Three of these target structures probably are different from already-described HLA-A2-restricted melanoma-associated antigens, because their expression in the different melanoma cell lines do not correlate with the recognition of melanoma cells by these CTL. The results first indicate that poorly immunogenic melanoma cells may express melanoma-associated antigens, and also suggest that, by using CTL clones obtained against different HLA-class-I-matched melanoma cells, it is possible to define such antigens.     

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and α₄β₁ integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and α₄β₁ integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and α₄β₁-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells. J. Cell. Physiol. 177:299–312, 1998. © 1998 Wiley-Liss, Inc.     

Cellular Stress Causes Accumulation of the Glucose Transporter at the Surface of Cells Independently of their Insulin Sensitivity

The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated 3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl) benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with an increase in the amount of the transporter on the cell surface. Cells subjected to K⁺-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected to K⁺ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J 4:1634–1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules at the cell surface. Received: 20 June 1995/Revised: 29 September 1995     

Secretion rates and levels of vascular endothelial growth factor in clone A or HCT-8 human colon tumour cells as a function of oxygen concentration

Molecular and in situ hybridization studies have shown, in a number of cell types, that under hypoxic conditions, vascular endothelial growth factor (VEGF) mRNA expression is up-regulated and VEGF protein is concomitantly increased. To establish a quantitative relationship between VEGF protein levels and oxygenation, we exposed exponentially growing clone A or HCT-8 human colon tumour cells in vitro (22 h at 37°C) to oxygen concentrations from 21% (air mixture) to 0.01%. Protein levels in cells and medium were then assayed using an enzyme-linked immunoabsorbent assay (ELISA). Intracellular levels of VEGF in clone A or HCT-8 cells exposed to either air (21% O₂) or the 0.01% O₂ mixture respectively increased from about 73 to 1270, and 1.5 to 1180 pg/10⁶ cells (about 17- and 80-fold increases). The shapes of the response curves (log of the intracellular VEGF concentrations v. log oxygen concentration) for both cell types were sigmoidal. However, intracellular VEGF levels in HCT-8 cells were always less than that of clone A cells until levels of about 0.3 to 0.1% O₂ were reached. Levels of VEGF in the supernatant were also increased after the 22 h hypoxic exposures. Because cell proliferation and clonogenicity were also measured, it was possible to estimate the secretion rates of VEGF for both cell lines as a function of oxygen percentage. For clone A cells, the secretion rate (pg/10⁶ cells/h) in 21% O₂ was 62.5. This rate increased to 428.8 pg/10⁶ cells/h at 0.01% O₂, a 7-fold increase. For HCT-8 cells, levels in the medium at 21% O₂ were too low to be measured by ELISA. However, between 10% and 0.01% O₂, secretion rates increased from 5.0 to 376.0 pg/10⁶ cells/h, a 75-fold increase. Therefore, at very low O₂ levels, VEGF secretion rates were similar in the two cell lines. We propose that the different VEGF responses of clone A and HCT-8 colon tumour cells to hypoxic stress in vitro are related to the in vivo observation that the respective hypoxic percentages of solid neoplasms originating from these cell lines are markedly different (i.e. about 3 versus 80%) at equivalent volumes of 750 mm³.     

Activation of the NLRP3 Inflammasome Complex is Not Required for Stress-Induced Death of Pancreatic Islets

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H₂O_2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.     

Glucose Deprivation Induces G2/M Transition-Arrest and Cell Death in N-GlcNAc2-Modified Protein-Producing Renal Carcinoma Cells

Some cancer cells can survive under glucose deprivation within the microenvironment of a tumor. Recently, we reported that N-linked (β-N-acetylglucosamine)₂ [N-GlcNAc₂]-modified proteins induce G2/M arrest and cell death under glucose deprivation. Here, we investigated whether such a response to glucose deprivation contributes to the survival of renal cell carcinomas, which are sensitive to nutritional stress. Specifically, we analyzed seven renal carcinoma cell lines. Four of these cell lines produced N-GlcNAc₂-modified proteins and led G2/M-phase arrest under glucose deprivation, leading to cell death. The remaining three cell lines did not produce N-GlcNAc₂-modified proteins and undergo G1/S-phase arrest under glucose deprivation, leading to survival. The four dead cell lines displayed significant up-regulation in the UDP-GlcNAc biosynthesis pathway as well as increased phosphorylation of p53, which was not observed in the surviving three cell lines. In addition, the four dead cell lines showed prolonged up-regulated expression of ATF3, which is related to unfolded protein response (UPR), while the surviving three cell lines showed only transient up-regulation of ATF3. In this study, we demonstrated that the renal carcinoma cells which accumulate N-GlcNAc₂-modified proteins under glucose deprivation do not survive with abnormaly prolonged UPR pathway. By contrast, renal carcinoma cells that do not accumulate N-GlcNAc₂-modified proteins under these conditions survive. Morover, we demonstrated that buformin, a UPR inhibitor, efficiently reduced cell survival under conditions of glucose deprivation for both sensitive and resistant phenotypes. Further studies to clarify these findings will lead to the development of novel chemotherapeutic treatments for renal cancer.