Endogenously Expressed Truncated P2X7 Receptor Lacking the C-Terminus is Preferentially Upregulated in Epithelial Cancer Cells and Fails to Mediate Ligand-Induced Pore Formation and Apoptosis

A truncated naturally occurring variant of the human purinergic receptor P2X7 (P2X7-R) was found in human cancer cervical cells. The novel protein consists of 258 amino acids, and compared to the wild-type P2X7-R it lacks the entire intracellular carboxy terminus, the second transmembrane domain, and the distal third of the extracellular loop. The truncated P2X7-R failed to form pores and mediate apoptosis, and it interacted with the wild-type P2X7-R in a manner suggesting auto-hetero-oligomerization. In contrast to cancer cells the novel truncated P2X7-R was expressed relatively little in normal cervical cells. These data raise the possibility that coexpression of the truncated form could block P2X7 mediated apoptosis and promote uncontrolled growth of cells.     

A truncated P2X7 receptor variant (P2X7-j) endogenously expressed in cervical cancer cells antagonizes the full-length P2X7 receptor through hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but full-length P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.     

Apically sorted P2X7 receptors mediate purinergic-induced pore formation preferentially in apical domains of the plasma membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.     

Apically Sorted P2X7 Receptors Mediate Purinergic-Induced Pore Formation Preferentially in Apical Domains of the Plasma Membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.     

Rod and cone pathway signalling is altered in the P2X7 receptor knock out mouse

The P2X7 receptor (P2X7-R) is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO) mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.     

Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.     

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.     

3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1,2,4] triazolo [3,4-b][1,3,4] thiadiazine,a novel tubulin inhibitor,evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells

Gastric cancer and cervical cancer are two major malignant tumors that threaten human health. The novel chemotherapeutic drugs are needed urgently to treat gastric cancer and cervical cancer with high anticancer activity and metabolic stability. Previously we have reported the synthesis, characterization and identification of a novel combretastatin A-4 analog, 3-(3-methoxyphenyl)-6-(3-amino-4- methoxyphenyl) -7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine (XSD-7). In this study, we sought to investigate its anticancer mechanisms in a human gastric cancer cell line (SGC-7901 cells) and human cervical carcinoma cell line (HeLa cells). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that XSD-7 induced cytotoxicity in SGC-7901 and HeLa cells with inhibitory concentration 50 values of 0.11 ± 0.03 and 0.12 ± 0.05 µM, respectively. Immunofluorescence studies proved that XSD-7 inhibited microtubule polymerization during cell division in SGC-7901 and HeLa cells. Then, these cells were arrested at G2/M cell cycle and subsequently progressed into apoptosis. In further study, mitochondrial membrane potential analysis and Western blot analysis demonstrated that XSD-7 treatment-induced SGC-7901 cell apoptosis via both the mitochondria-mediated pathway and the death receptor-mediated pathway. In contrast, XSD-7 induced apoptosis in HeLa cells mainly via the mitochondria-mediated pathway. Hence, our data indicate that XSD-7 exerted antiproliferative activity by disrupting microtubule dynamics, leading to cell cycle arrest, and eventually inducing cell apoptosis. XSD-7 with novel structure has the potential to be developed for therapeutic treatment of gastric cancer and cervical cancer.     

Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.     

Regulation of the P2X7R by microRNA-216b in human breast cancer

Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that P2X7R was up-regulated and miR-216b was down-regulated in breast cancer cell lines and tissues. Using bioinformatic analysis and 3′UTR luciferase reporter assay, we determined P2X7R can be directly targeted by miR-216b, which can down-regulate endogenous P2X7R mRNA and protein levels. Ectopic expression of miR-216b mimics leads to inhibited cell growth and apoptosis, while blocking expression of the miR-216b results in increased cell proliferation. Furthermore, our findings demonstrate that knockdown of P2X7R promotes apoptosis in breast cancer cells through down-regulating Bcl-2 and increasing the cleavage caspase-3 protein level. Finally, we confirmed that down-regulation of miR-216b in breast cancer is inversely associated with P2X7R expression level. Together, these findings establish miR-216b as a novel regulator of P2X7R and a potential therapeutic target for breast cancer.     

Down‐regulation of p16 and MGMT promotes the anti‐proliferative and pro‐apoptotic effects of 5‐Aza‐dC and radiation on cervical cancer cells

Cervical cancer is one of the most common malignancies of the female reproductive system. Therefore, it is critical to investigate the molecular mechanisms involved in the development and progression of cervical cancer. In this study, we stimulated cervical cancer cells with 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC) and found that this treatment inhibited cell proliferation and induced apoptosis; additionally, methylation of p16 and O‐6‐methylguanine‐DNA methyltransferase (MGMT) was reversed, although their expression was suppressed. 5‐Aza‐dC inhibited E6 and E7 expression and up‐regulated p53, p21, and Rb expression. Cells transfected with siRNAs targeting p16 and MGMT as well as cells stimulated with 5‐Aza‐dC were arrested in S phase, and the expression of p53, p21, and Rb was up‐regulated more significantly. However, when cells were stimulated with 5‐Aza‐dC after transfection with siRNAs targeting p16 and MGMT, proliferation decreased significantly, and the percentage of cells in the sub‐G1 peak and in S phase was significantly increased, suggesting a marked increase in apoptosis. But E6 and E7 overexpression could rescue the observed effects in proliferation. Furthermore, X‐ray radiation caused cells to arrest in G2/M phase, but cells transfected with p16‐ and MGMT‐targeted siRNAs followed by X‐ray radiation exhibited a significant decrease in proliferation and were shifted toward the sub‐G1 peak, also indicating enhanced apoptosis. In addition, the effects of 5‐Aza‐dC and X‐ray radiation were most pronounced when MGMT expression was down‐regulated. Therefore, down‐regulation of p16 and MGMT expression enhances the anti‐proliferative effects of 5‐Aza‐dC and X‐ray radiation. This discovery may provide novel ideas for the treatment of cervical cancer.     

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca²⁺ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.     

Reversal of multidrug resistance in vitro and in vivo by 5-N-formylardeemin,a new ardeemin derivative

Because multidrug resistance (MDR) is a serious impediment to the use of chemotherapy in treating cancer patients, great efforts have been made to search for effective MDR-reversing agents. We have developed a brand new synthetic ardeemin derivative, 5-N-formylardeemin, and investigated the activity of which in reversing MDR in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin, vincristine through potentiation of apoptosis in both MCF-7-R and A549-R at relatively noncytotoxic concentrations in vitro. Mechanistic studies showed that 5-N-formylardeemin inhibited the expression of MDR-1 (P-gp) and increased the intracellular accumulation of cytotoxic drugs in the MDR cells, suggesting that 5-N-formylardeemin reverses MDR activities through inhibiting MDR-1 expression. Interestingly, 5-N-formylardeemin also sensitized the parent wild-type cancer cells toward these chemotherapeutic agents to various extents. Importantly, in vivo studies demonstrated that 5-N-formylardeemin significantly improved the therapeutic effects of doxorubicin in nude mice bearing A549-R xenografts, which was associated with reduced expression of MDR-1 protein level and increased apoptosis in tumor tissues. These results underscore 5-N-formylardeemin as a potential sensitizer for chemotherapy against multidrug resistant cancers.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.     

Caspase cleavage enhances the apoptosis-inducing effects of BAD

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.     

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.