Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats.

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.

Effect of caesarean section on maternal and foetal outcomes in acute fatty liver of pregnancy: a systematic review and meta-analysis.

Several studies have reported a positive association between caesarean section for expeditious pregnancy termination and perinatal outcomes in acute fatty liver of pregnancy (AFLP); however, the risks remain unclear and independent studies have reported conflicting findings. In this meta-analysis, we aimed to confirm the relationship between caesarean section and perinatal outcomes in AFLP. The PubMed, Embase, and China National Knowledge Infrastructure databases were searched (until July 17, 2015) for observational clinical studies focusing on the association between caesarean section and perinatal outcomes in AFLP. Data were extracted and processed independently by 2 authors. We also compared caesarean section with vaginal delivery to further investigate this relationship. We observed that 2 of the 3 primary outcomes in caesarean section exhibited positive effects-the maternal mortality rate was 44% lower (relative risk [RR], 0.56 [0.41-0.76]) and perinatal mortality rate was also reduced (RR, 0.52 [0.38-0.71]), compared to those for vaginal delivery. We did not find any associations between caesarean section and perinatal outcomes in AFLP in terms of neonatal mortality type and maternal multiple organ complications. These findings emphasise the significant prognostic value and clinical implications of caesarean section in AFLP, and suggest that the adverse outcomes should be reduced.

Innate immune cell-derived microparticles facilitate hepatocarcinoma metastasis by transferring integrin α(M)ß2 to tumor cells.

Mechanisms by which tumor cells metastasize to distant organs still remain enigmatic. Immune cells have been assumed to be the root of metastasis by their fusing with tumor cells. This fusion theory, although interpreting tumor metastasis analogically and intriguingly, is arguable to date. We show in this study an alternative explanation by immune cell-derived microparticles (MPs). Upon stimulation by PMA or tumor cell-derived supernatants, immune cells released membrane-based MPs, which were taken up by H22 tumor cells, leading to tumor cell migration in vitro and metastasis in vivo. The underlying molecular basis was involved in integrin α(M)ß2 (CD11b/CD18), which could be effectively relayed from stimulated innate immune cells to MPs, then to tumor cells. Blocking either CD11b or CD18 led to significant decreases in MP-mediated tumor cell metastasis. This MP-mediated transfer of immune phenotype to tumor cells might also occur in vivo. These findings suggest that tumor cells may usurp innate immune cell phenotypes via MP pathway for their metastasis, providing new insight into tumor metastatic mechanism.

Expression of cytokines in mouse hepatitis B virus X gene-transfected model.

The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

Delivery of chemotherapeutic drugs in tumour cell-derived microparticles.

Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

New alternatively spliced variant of prostate-specific membrane antigen PSM-E suppresses the proliferation, migration and invasiveness of prostate cancer cells.

PSM-E is a newly discovered alternatively spliced variant of prostate-specific membrane antigen (PSMA). In the current study, its role on the proliferation, invasiveness and migration in prostate cancer cell lines was analyzed. PSM-E and PSMA (as a comparison) eukaryotic expression vectors pcDNA3.0/PSM-E and pcDNA3.0/PSMA were constructed, validated by RT-PCR and Western blotting, and PSMA/PSM-E overexpression PC-3 cell models were built. Gene interference was used to block PSMA and the expression of its splice variants in LNCap cells. Three shRNA fragments were synthesized against PSMA, cloned into the vector pSilencer 2.1-U6-neo, their interference effect was evaluated by RT-PCR and Western blotting, and pSilencer 2.1-U6-neo­shRNA3 (named p­shRNA3) was chosen in further analyses. Growth curves were drawn to observe the proliferation change, which showed that PSM-E had the potential to suppress proliferation (P<0.05), but no significant change was observed in PSMA/PC-3 cells and in PSMA/PSM-E interfering LNCap cells (P>0.05). Cross-river test showed that the migration speeds of PSM-E/PC-3 and PSMA/PC-3 were both significantly slower than the vector negative control, and faster in p-shRNA3 interfering LNCap cells compared with its vector negative control (P<0.05), and no significant difference existed between PSM-E/PC-3 and PSMA/PC-3 (P>0.05). Transwell assay showed that the invasive cells of both PSMA/PC-3 and PSM-E/PC-3 were fewer compared to the vector negative control (P<0.05), and the invasive suppression effect of PSM-E was weaker than PSMA (P<0.05), and accordingly, invasiveness of interfering LNCaP cells was enhanced compared with the vector negative control (P<0.05). These results showed that PSM-E could suppress proliferation, migration and invasiveness of prostate cancer cells. Its suppression effect on cell proliferation is stronger compared to PSMA and the suppression effect on invasiveness is weaker than that of PSMA.

Depletion of regulatory T cells facilitates growth of established tumors: a mechanism involving the regulation of myeloid-derived suppressor cells by lipoxin A4.

Regulatory T cells (Tregs) are thought to facilitate tumor development by suppressing protective antitumor immune responses. However, recent clinical and laboratory studies show that Tregs are a favorable element against cancer. In this study, we provide evidence that Tregs have both promoting and inhibiting effects on tumors, depending on the stage of tumor development. By using 0.5 mg cyclophosphamide, we constructed a murine liver cancer model in which Tregs were continuously and selectively depleted. Under such conditions, we found that tumor growth was inhibited at early stages but accelerated later on. Analysis of the tumor microenvironment disclosed that long-term Treg depletion by 0.5 mg cyclophosphamide treatment induced Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Ablation of MDSCs by anti-Gr-1 Ab blocked Treg depletion-induced promotion of tumor growth. Furthermore, lipoxygenases 5 and 12, two enzymes participating in the biosynthesis of the lipid anti-inflammatory mediator lipoxin A(4), were upregulated or downregulated by Treg depletion or adoptive transfer. Correspondingly, the levels of lipoxin A(4) were increased or decreased. Lipoxin A(4) thus regulated the induction of MDSCs in response to Treg depletion. These findings suggest that Tregs may play different roles at different stages of tumor growth: promoting early and inhibiting late tumor growth. Our study also suggests that the interplay among Tregs, MDSCs, and lipoxin A(4) tunes the regulation of tumor-associated inflammation.

Microparticles mediate enzyme transfer from platelets to mast cells: a new pathway for lipoxin A4 biosynthesis.

The inflammation-resolving lipid mediator lipoxin A4 (LXA4), which is derived from arachidonic acid in the context of inflammation, can be generated physiologically in vivo. However, the mechanism of physiologic formation of LXA4 remains elusive. In this report, we provide evidence that platelet-derived microparticles contain lipoxygenase 12 (12-LO) protein and act as a mediator in transferring 12-LO to mast cells, leading to the production of LXA4 by mast cells. Absence of either leukotriene, the precursor for LXA4, in mast cells or 12-LO in microparticles abolished LXA4 production. Using a mouse model, we demonstrated that platelet-derived microparticles were taken up by peritoneal mast cells in vivo and triggered LXA4 production. We also found that similar to LXA4, platelet-derived microparticles attenuated LPS- or dextran sulfate sodium-induced inflammation by regulating inflammatory cytokines. Together, these data suggest a critical role of platetlet-derived microparticles as a signal mediator, at least in LXA4 production, resulting in significant immunoregulatory consequences.

CD4+FOXP3+ regulatory T cell depletion by low-dose cyclophosphamide prevents recurrence in patients with large condylomata acuminata after laser therapy.

Condylomata acuminata (CA) caused by human papillomavirus (HPV) is a common sexually transmitted disease with half a million new cases diagnosed in the United States per year and the annual increase in incidence in China. Recurrence is a major challenge for CA treatment. Recently, we demonstrated that FOXP3(+) regulatory T (Treg) cells mediate the immunosuppression in large genital warts. Here, we further report that low-dose cyclophosphamide (CY), a conventional chemotherapy drug, can effectively prevent the recurrence of large CA in clinical patients after laser therapy. Surprisingly, although 9 out of 52 patients recur six weeks after the combination treatment, the re-administration of low-dose CY alone completely eliminates most recurred lesions. We provide evidence that low-dose CY not only depletes patients' Treg cells and enhances function of HPV-specific T cells and NK cells in the periphery, but also ameliorates the immune milieu of the lesion site, leading to the elimination of remnant viruses. These findings have important clinical significance, and potentially lead to a therapeutic breakthrough for the treatment of CA.

Hepatitis B virus protein X-induced expression of the CXC chemokine IP-10 is mediated through activation of NF-kappaB and increases migration of leukocytes.

Interferon-gamma inducible protein 10 (IP-10) involves inflammatory cell recruitment and cellular immune damage during virus infection. Although an increase of the peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. Transfection of the HBx-expressing vector into HepG2 cells results in nuclear translocation of NF-kappaB, which directly binds the promoter of IP-10 at positions from -122 to -113, thus facilitating transcription. The addition of the NF-kappaB inhibitor blocks the effect of HBx on IP-10 induction. In parallel, increase of NF-kappaB subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces activation of NF-kappaB through the TRAF2/TAK1 signaling pathway, leading to up-regulation of IP-10 expression. As a consequence, up-regulation of IP-10 may mediate the migration of peripheral blood leukocytes in a NF-kappaB-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-kappaB pathway, leading to transactivation of the IP-10 promoter. Our study provides insight into the migration of leukocytes in response to HBV infection, thus causing immune pathological injury of liver.

Transforming growth factor-beta1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells.

AIM: To investigate whether rhTGF-beta1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-beta receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells. METHODS: MCF-7 breast cancer cells were treated with rhTGF-beta1 or transfected with a recombinant vector, pIRES2-EGFP-TbetaRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1alpha. RESULTS: CXCR4 mRNA and protein expression were upregulated in TGF-beta1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1alpha. In MCF-7 cells transiently transfected with pIRES2-EGFP-TbetaRII-Fc, a fusion protein named TbetaRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1alpha-mediated chemotactic response. CONCLUSION: TGF-beta1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1alpha-induced chemotactic response. The results suggest a link between TGF-beta1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-beta1-mediated enhancement of metastatic potential in breast cancer cells.

Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model.

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.

Rapamycin inhibits lung metastasis of B16 melanoma cells through down-regulating alphav integrin expression and up-regulating apoptosis signaling.

Currently available data indicate the potential application of rapamycin and its analogues in the clinic as anticancer therapeutic agents through inhibiting tumor cell growth and tumor angiogenesis. However, whether rapamycin can directly suppress tumor metastasis remains unclear. In the present study, we demonstrated that rapamycin treatment results in reduced formation of metastatic nodules in the lung by B16 cells. This is due to two mechanisms. First, the expression of alphav integrin is down-regulated by rapamycin treatment, and subsequently, the phosphorylation of focal adhesion kinase (FAK) is reduced. Second, rapamycin promotes apoptosis by up-regulating the proapoptotic molecules Bid and Bax and down-regulating Bcl-xL. Blocking the apoptosis pathway by pan-caspase inhibitor zVAD partially reversed the suppression of rapamycin in B16 metastasis. Interestingly, rapamycin up-regulates Bax and Bid in B16 cells via the S6K1 pathway and down-regulates the expression of alphav integrin via other pathway(s). In addition, our data showed that autophagy was not involved in the mechanisms of rapamycin-mediated metastasis suppression. Our findings demonstrate a potential anti-metastatic effect of rapamycin via down-regulating alphav integrin expression and up-regulating apoptosis signaling, suggesting that rapamycin might be worthy of clinical evaluation as an antimetastatic agent.

Engineering and characterization of a baculovirus-expressed mouse/human chimeric antibody against transferrin receptor.

Transferrin receptor (TfR) has been explored as a target for antibody-based therapy of cancer. In the previous study, we reported a murine anti-TfR monoclonal antibody (mAb) 7579 had good anti-tumor activities in vitro. In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effector mechanism in vivo, we herein developed its chimera in the baculovirus/insect cell expression system based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The chimeric light and heavy chains, containing human IgG1 constant regions, were correctly processed and assembled in insect cells, and then secreted into the mediums as heterodimeric H(2)L(2) immunoglobulins. Furthermore, analyses of antigen-binding assay and competitive binding assay indicated that the chimeric antibody possessed specificity and affinity similar to that of its parental murine antibody. Results of the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assay verified that the chimeric antibody could efficiently mediate ADCC and CDC against TfR-overexpressing tumor cells. These results suggested that this baculovirus-expressed chimeric anti-TfR IgG1 might have the potential to be used for cancer immunotherapy. Meanwhile, the MAGIC strategy, facilitating the rapid generation of chimeric mAbs, could be one of the efficient strategies for antibody engineering.

Arsenic trioxide downregulates the expression of annexin II in bone marrow cells from patients with acute myelogenous leukemia.

BACKGROUND: Most patients with acute myelogenous leukemia (AML) suffer from disordered hemostasis. We have previously shown that annexin II (Ann II), a high-affinity co-receptor for plasminogen/tissue plasminogen activator, plays a central role in primary hyperfibrinolysis in patients with acute promyelocytic leukemia (APL). The expression of Ann II in cells from patients with major subtypes of AML and the effect of arsenic trioxide (As2O3) on Ann II expression in AML cells were investigated to determine whether As2O3-mediated downregulation of Ann II could restore hemostatic stability. METHODS: A total of 103 patients (48 females and 55 males; age, 19 - 58 years) were included. Plasma samples were collected before and after treatment as well as after complete remission. Ann II and plasminogen activation were measured in leukemic cells during treatment with 1 micromol/L As2O3. RESULTS: Before As2O3 treatment, Ann II mRNA expression (real-time PCR) was the highest in M3 cells (P < 0.05), higher in M5 cells than that in M1, M2, M4, and M6 cells (P < 0.001), and positively correlated with Ann II protein expression (flow cytometry) (r = 0.752, P < 0.01). Exposure for up to 120 hours to As2O3 (1 micromol/L) had no significant effect on Ann II protein in M1 and M2 leukemic cells, but decreased Ann II protein expression twofold within 48 hours of exposure in M3 cells (P < 0.05) and twofold within 96 hours in M5 cells (P < 0.05). The rate of plasmin generation was higher in APL, M5, and M4 cells than in M1, M2, and M6 cells. CONCLUSIONS: As2O3 may reduce hyperfibrinolysis in AML by downregulation of Ann II. Furthermore, As2O3 affects more than one form of AML (APL, M4 and M5), suggesting its potential role in their management.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AIM: Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatin's (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved. METHODS: SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT. RESULTS: The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G(1)/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells. CONCLUSION: Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G(1)/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.Acta Pharmacologica Sinica (2009) 30: 1053-1059; doi: 10.1038/aps.2009.59.

HBx protein induces expression of MIG and increases migration of leukocytes through activation of NF-kappaB.

Elevated expression of monokine induced by the interferon-gamma (MIG) has been shown in HBV carriers, and it is involved in the infiltration of inflammatory cells and liver damage after HBV infection. However, the molecular mechanisms by which HBV-induced MIG expression have not been characterized. Our results indicated that HBx protein induced MIG expression in a dose-dependent manner. Such increase was due to the direct binding of NF-kappaB to the MIG promoter. By luciferase, chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that the NF-kappaB binding site at positions -147 was essential for transcriptional activation of MIG promoter by HBx protein. Chemotaxis assay showed that the up-regulation of MIG protein levels enhanced the migration of peripheral blood lymphocytes (PBLs), and inhibition of NF-kappaB significantly decreased the chemotaxis activity. Our findings provide a new insight into how leukocytes migrate to liver, and disclose a new regulatory mechanism of MIG expression after HBV infection.

miR-142-3p restricts cAMP production in CD4+CD25- T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4(+)CD25(+) regulatory T (T(REG)) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR-142-3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4(+)CD25(-) T cells and CD4(+)CD25(+) T(REG) cells. miR-142-3p limits the level of cAMP in CD4(+)CD25(-) T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR-142-3p to keep the AC9/cAMP pathway active in CD4(+)CD25(+) T(REG) cells. These findings reveal a new molecular mechanism through which CD4(+)CD25(+) T(REG) cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.

Transcriptional upregulation of HSP70-2 by HIF-1 in cancer cells in response to hypoxia.

Heat shock protein 70-2 (HSP70-2) can be expressed by cancer cells and act as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate HSP70-2 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the expression of HSP70-2 had a significant increase in cancer cells. Such increase was due to the direct binding of hypoxia-inducible factor to hypoxia-responsive elements (HREs) in the HSP70-2 promoter. By luciferase assays, we demonstrated that the HRE1 at position -446 was essential for transcriptional activation of HSP70-2 promoter under hypoxic conditions. We also demonstrated that HIF-1alpha binds to the HSP70-2 promoter and the binding is specific, as revealed by HIF binding/competition and chromatin immunoprecipitation assays. Consequently, the upregulation of HSP70-2 enhanced the resistance of tumor cells to hypoxia-induced apoptosis. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of HSP70-2 expression in tumor cells.