Deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins in hyper-radiosensitive Long-Evans Cinnamon (LEC) rat cells after X irradiation

The DNA-dependent protein kinase (DNA-PK) complex has been implicated in the repair of DNA double-strand breaks (DSBs). DNA-PK is a heterotrimeric protein complex comprised of two components: a large catalytic subunit, Prkdc, with serine/threonine kinase activity and a DNA-targeting component, G22p1 and Xrcc5. In previous report, we showed that approximately 80% of the G22p1 and Xrcc5 proteins were observed in the cytoplasm of rat fibroblasts, and that nuclear translocation of the proteins from the cytoplasm is important for the repair of DNA DSBs. In the present study, we showed that nuclear accumulation of the G22p1 and Xrcc5 proteins was not observed in fibroblasts from a mutant strain of Long-Evans Cinnamon (LEC) rat that has an enhanced radiosensitivity and a reduced level of repair of DSBs after X irradiation. Nuclear translocation of the proteins was observed in both LEC rat cells and control rat cells with normal radiosensitivity at 5 min after X irradiation. Although high levels of G22p1 and Xrcc5 proteins were observed in the nuclei of control rat cells until 60 min postirradiation, the amounts of the proteins decreased rapidly in the nuclei of LEC rat cells in the first 10 min after X irradiation. These findings suggest that there are some defects in maintaining the levels of G22p1 and Xrcc5 proteins in the nuclei of LEC rat cells. An analysis of fibroblasts from backcross rats showed that the deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins is genetically linked to enhanced radiosensitivity. Since the nucleotide sequences of the G22p1 and Xrcc5 genes of the LEC rats coincided with those of the control rats, the deficiency in nuclear accumulation may not be caused by mutations of the G22p1 and Xrcc5 proteins.     

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.     

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.     

Involvement of the nonhomologous end joining DNA repair pathway in the bystander effect for chromosomal aberrations

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.     

Expression and chromosome location of hamster Ku70 and Ku80

Ku proteins play an important role in DNA double-strand break (DSB) repair, chromosome maintenance, and growth regulation. To understand the fundamental characteristics of Ku proteins, we examined the electrophoretic mobility and expression of hamster Ku70 and Ku80 and determined the chromosome locations of their genes. The electrophoretic mobility of hamster Ku proteins are different from that of human Ku proteins. No significant changes in the quantity of Ku proteins were observed in CHO-K1 cells treated with 10 Gy of ionizing radiation, suggesting that both proteins are expressed constitutively in amounts adequate to repair DNA DSBs. The chromosome locations of the Ku genes were determined by direct R-banding fluorescence in situ hybridization. The Ku70 gene was localized to Syrian hamster chromosome 4qa4.1--> qa4.2 and Chinese hamster chromosome 2p3.1, and the Ku80 gene was localized to Syrian hamster chromosome 4qb5--> qb6.1 and Chinese hamster chromosome 2p3.5-->p3.6. These results provide clues to the biological functions of Ku, as well as useful information for constructing comparative chromosome maps between hamsters and other mammalian species, including human, mouse, and rat.     

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.     

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.     

In Vivo Association of Ku with Mammalian Origins of DNA Replication

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.     

沉默NHEJ修复通路中关键蛋白Ku70在牙髓干细胞增殖和凋亡中的作用及其机制研究

目的:研究沉默非同源重组修复(non-homologous endjoining,MHEJ)通路中关键蛋白Ku70在牙髓干细胞增殖和凋亡中的作用,分析其机制.方法:提取健康恒牙牙髓组织,进行牙髓干细胞培养.采用脂多糖诱导人牙髓干细胞,分为对照组、阴性对照组、脂多糖组、沉默组和沉默+脂多糖组.观察Ku70免疫组化情况,进...     

Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK

DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.     

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.     

Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength.

Proteins were extracted from isolated rat liver nuclei with 0.15 M NaCl and 0.35 M NaCl at pH 8.0. The number of phosphoproteins in these extracts was determined by labeling with 32P and autoradiography after two-dimensional gel electrophoresis. Two proteins, B22p and B24p, contained small amounts of 32P and sedimented with the 30S nuclear informofer particle. With the exception of two phosphoproteins, CB and CN', all of the phosphoproteins found in the 0.35 M NaCl extract. Approximately 20% of the 0.15 M NaCl soluble proteins bound to rat liver DNA in 0.05 M KCl-0.05 M Tris-HCl (pH 8). Of these proteins, 1-2% bound to DNA in 0.15 M KCl and were eluted with 2 M KCl. This DNA bound fraction which contained both phosphorylated and nonphosphorylated proteins was similar in both the 0.15 and 0.35 M NaCl extracts. However, two major proteins (C13 and C14) and three minor proteins (C15, C25, Cg') were present only in the 0.15 M NaCl extract. The results of the present study show that there are marked similarities in the two-dimensional gel electrophoretic, phosphorylation, and DNA binding properties of rat liver nuclear proteins soluble in either 0.15 or 0.35 M NaCl.     

A comparison of chromosome repair kinetics in G(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts for increased potentially lethal damage repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.     

Interaction of a cyclin E fragment with Ku70 regulates Bax-mediated apoptosis

The cyclin E/Cdk2 complex plays an essential role in the G(1)/S cell cycle transition and DNA replication. Earlier we showed that in hematopoietic tumor cells, caspase-mediated cleavage of cyclin E generates p18-cyclin E, which is unable to interact with Cdk2 and therefore plays a role independent of the cell cycle. The expression of a cleavage-resistant cyclin E mutant greatly diminishes apoptosis, indicating the critical role of cyclin E cleavage. p18-cyclin E expression can induce apoptosis or sensitization to apoptotic stimuli in many cell types. Here we identify Ku70 as a specific p18-cyclin E-interacting partner. In hematopoietic tumor cell lines, the association of p18-cyclin E with Ku70 induces the dissociation of Bax from Ku70, followed by Bax activation. This mechanism of Bax activation leads to the amplification of the apoptosis signal in all tumor cell lines examined. N-terminal Ku70 deletion mutants are unable to bind to p18-cyclin E to regulate its apoptotic effect. p18-cyclin E-mediated amplification of apoptosis is dependent on Bax and Ku70 being greatly diminished in Ku70(-/-) and Bax(-/-) mouse embryo fibroblasts and in hematopoietic cells where Bax knockdown was achieved by short interfering RNA. The p18-cyclin E/Ku70 and Bax/Ku70 interactions provide a balance between apoptosis and the survival of cells exposed to genotoxic stress.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence

Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome. The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L. A.(1999) J. Biol. Chem. 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M. (2000) J. Biol. Chem. 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation. We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins. The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity. The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1. 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences. However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter). Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42. Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing.     

Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species.

The Ku (p70/p80) autoantigen is a DNA-protein complex recognized by sera from certain patients with SLE and related diseases. Although human autoantibodies react with at least eight different epitopes of the human Ku complex, they had little reactivity with rodent Ku Ag on immunoblots. Small amounts of 70- and 80-kDa proteins were immunoprecipitated from murine cell extracts, however, suggesting that the Ku particle is not unique to human cells. This was confirmed by isolating cDNA clones encoding murine Ku Ag by plaque hybridization with a human p70 cDNA probe. The murine p70 cDNA clones had a deduced amino acid sequence 82.9% identical to that of human p70, and comparable amounts of murine and human p70 mRNA were detected in 3T3 and K562 cells, respectively. The poor reactivity of human autoantibodies with murine p70 was attributable to specific amino acid substitutions in an immunodominant conformational epitope located on amino acids 560-609 of human p70. Several amino acids critical for antigenicity of this region were defined by mutagenesis studies. Other conformational epitopes of Ku were also antigenically poorly conserved among species. Species-specific epitopes recognized by lupus autoantibodies are unusual but not unique to Ku. In general, poorly conserved autoepitopes have been conformational, rather than sequential, suggesting that the antigenicity of conformational epitopes may be particularly sensitive to evolutionary change.     

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.