DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-SIN1 association mediates ultraviolet B (UVB)-induced Akt Ser-473 phosphorylation and skin cell survival.

BACKGROUND: The exposure of skin keratinocytes to Ultraviolet (UV) irradiation leads to Akt phosphorylation at Ser-473, which is important for the carcinogenic effects of excessive sun exposure. The present study investigated the underlying mechanism of Akt Ser-473 phosphorylation by UVB radiation. RESULTS: We found that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mammalian target of rapamycin (mTOR) complex 2 (mTORC2) were both required for UVB-induced Akt Ser-473 phosphorylation in keratinocytes. Inhibition of DNA-PKcs activity via its inhibitor NU7026, a dominant-negative kinase-dead mutation, RNA interference (RNAi) or gene depletion led to the attenuation of UVB-induced Akt Ser-473 phosphorylation. Meanwhile, siRNA silencing or gene depletion of SIN1, a key component of mTORC2, abolished Akt Ser-473 phosphorylation by UVB. Significantly, we discovered that DNA-PKcs was associated with SIN1 in cytosol upon UVB radiation, and this complexation appeared required for Akt Ser-473 phosphorylation. Meanwhile, this DNA-PKcs-SIN1 complexation by UVB was dependent on epidermal growth factor receptor (EGFR) activation, and was disrupted by an EGFR inhibitor (AG1478) or by EGFR depletion. UVB-induced complexation between DNA-PKcs and mTORC2 components was also abolished by NU7026 and DNA-PKcs mutation. Finally, we found that both DNA-PKcs and SIN1 were associated with apoptosis resistance of UVB radiation, and inhibition of them by NU7026 or genetic depletion significantly enhanced UVB-induced cell death and apoptosis. CONCLUSION: Taken together, these results strongly suggest that DNA-PKcs-mTORC2 association is required for UVB-induced Akt Ser-473 phosphorylation and cell survival, and might be important for tumor cell transformation.

Sphingosine kinase-1 inhibition sensitizes curcumin-induced growth inhibition and apoptosis in ovarian cancer cells.

Recent published studies suggest that increasing levels of ceramides enhance the chemo-sensitivity of curcumin. Using in vitro approaches, we analyzed the impact of sphingosine kinase-1 (SphK-1) inhibition on ceramide production, and evaluated SphK1 inhibitor II (SKI-II) as a potential curcumin chemo-sensitizer in ovarian cancer cells. We found that SphK1 is overexpressed in ovarian cancer patients' tumor tissues and in cultured ovarian cancer cell lines. Inhibition of SphK1 by SKI-II or by RNA interference (RNAi) knockdown dramatically enhanced curcumin-induced apoptosis and growth inhibition in ovarian cancer cells. SKI-II facilitated curcumin-induced ceramide production, p38 activation and Akt inhibition. Inhibition of p38 by the pharmacological inhibitor (SB 203580), a dominant-negative expression vector, or by RNAi diminished curcumin and SKI-II co-administration-induced ovarian cancer cell apoptosis. In addition, restoring Akt activation introducing a constitutively active Akt, or inhibiting ceramide production by fumonisin B1 also inhibited the curcumin plus SKI-II co-administration-induced in vitro anti-ovarian cancer effect, suggesting that ceramide accumulation, p38 activation and Akt inhibition are downstream effectors. Our findings suggest that low, well-tolerated doses of SKI-II may offer significant improvement to the clinical curcumin treatment of ovarian cancer.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway.

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.

Increasing ceramides sensitizes genistein-induced melanoma cell apoptosis and growth inhibition.

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects.

[Cross-talk between nuclear factor-kappaB and P53 signal pathway in keratinocytes after ultraviolet B irradiation].

OBJECTIVE: To investigate the cross-talk between nuclear factor kappaB (NF-kappaB) and P53 signal pathways in human epidermal keratinocytes (NHEKs) after ultraviolet B (UVB) irradiation. METHODS: Normal NHEKs harboring wild p53 gene and immortal human keratinocytes of the line HaCaT harboring mutant p53 gene were cultured at 37 degrees C in an atmosphere containing 5% CO(2) and then underwent irradiation of UVB of the dose of 60 mJ/cm(2). Part of the NHEKs and HaCaT cells were pretreated with BAY11-7082, NF-kappaB inhibitor inhibiting IkB-a phosphorylation, of the final concentration of 5 micromol/L. Western blotting was used to detect the protein expression of NF-kappaB, P53, and P21. The transcriptional activity of NF-kappaB was analyzed by electrophoretic mobility shift assay (EMSA). RESULTS: UVB irradiation increased the protein expression of NF-kappaB, P53, and P21 and triggered the transcriptional activity of NF-kappaB. BAY11-7082 significantly inhibited the NF-kappaB protein expression induced by UVB irradiation in the NHEKs and HaCaT cells. The P53 protein expression of the NHEKs undergoing pretreatment of BAY11-7082 and UVB irradiation was 0.08 +/- 0.07, significantly lower than that of the NHEKs undergoing only UVB irradiation (0.78 +/- 0.32, P < 0.01). The P21 protein expression of the NHEKs undergoing pretreatment of BAY11-7082 and UVB irradiation was 0.65 +/- 0.22, significantly lower than that of the NHEKs undergoing only UVB irradiation (1.58 +/- 0.77, P < 0.05). However, the P53 and the P21 protein expression of the HaCaT cells undergoing pretreatment of BAY11-7082 and UVB irradiation was not significantly different from that of the HaCaT cells undergoing only UVB irradiation. CONCLUSION: There exists a UVB-induced cross-talk between NF-kappaB and P53 signal pathways in human epidermal keratinocytes. This cross-talk is correlated with P53 functional condition.

[Effects of antisense epidermal growth factor receptor oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes].

OBJECTIVE: To explore the effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro. METHODS: c-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method. RESULTS: Compared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01). CONCLUSION: The ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.

MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

UVB-irradiated human keratinocytes and interleukin-1alpha indirectly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradiated dermal fibroblasts.

BACKGROUND: Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts. METHODS: Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts. CONCLUSIONS: UVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

Green tea polyphenol epigallocatechin-3-gallate inhibits the expression of nitric oxide synthase and generation of nitric oxide induced by ultraviolet B in HaCaT cells.

BACKGROUND: Nitic oxide (NO) has been implicated in the pathogenesis of various inflammatory diseases, including sunburn and pigmentation induced by ultraviolet irradiation. Epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can protect skin from ultraviolet-induced damage. The purpose of this study was to investigate the protective mechanisms of EGCG on inducible nitric oxide synthase (iNOS) expression and NO generation by ultraviolet B (UVB) irradiation in HaCaT cells. METHODS: HaCaT cells were irradiated with UVB 30 mJ/cm 2 and pretreated with EGCG at varying concentrations. The iNOS mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and NO production was quantified by spectrophotometric method. The expression of NF-kappaB P65 was measured by immunofluorescence cytochemistry staining. RESULTS: The expression of iNOS mRNA and generation of NO in HaCaT cells were increased by UVB irradiation. EGCG down regulated the UVB-induced iNOS mRNA synthesis and NO generation in a dose dependent manner. The UVB-induced ctivation and translocation of NF-kappaB were also down regulated by EGCG treatment in HaCaT cells (P < 0.01). CONCLUSIONS: Green tea derived-EGCG can inhibit and down regulate the UVB-induced activation and translocation of NF-kappaB, expression of iNOS mRNA and generation of NO respectively, indicating EGCG may play a protective role from UVB-induced skin damage.

An investigation of crosstalk between Wnt/ß-catenin and transforming growth factor-ß signaling in androgenetic alopecia.

BACKGROUND: Wnt and transforming growth factor-ß (TGF-ß) signaling pathways are known to be involved in the pathogenesis of androgenetic alopecia (AGA). However, the way that Wnt and TGF-ß signaling is altered in patients with AGA and whether there exists a crosstalk between them in pathogenetic process of AGA remain unclear. OBJECTIVES: To investigate the expression of Wnt and TGF-ß signaling and the crosstalk between these 2 signaling pathways in AGA. METHODS: Fifteen male patients with AGA were recruited for our research. Fifteen scalp specimens of the balding were collected from frontal areas, and 9 nonbalding were collected from occipital areas. We analyzed the expression and activation of downstream Wnt and TGF-ß signaling molecules in both balding and nonbalding hair follicles isolated from scalp specimens. Furthermore, we evaluated the activation of Wnt and TGF-ß signaling after either of them was blocked with the inhibitor in balding and nonbalding dermal papilla (DP) cells. RESULTS: Compared with the nonbalding counterparts, the mRNA level of Wnt10a and LEF1 was decreased. But TßRI and TßRII, and the protein expression of TGF-ß1 was elevated in balding hair follicles. To investigate the crosstalk between Wnt and TGF-ß signaling, we used SB431542 to inhibit the TGF-ß signaling in balding DP cells and found that SB431542 significantly attenuated the phosphorylation of Smad2 and Akt. However, the mRNA level of Wnt10a, LEF1, and the nuclear translocation of ß-catenin was increased. On the other hand, we suppressed the Wnt signaling by XAV939 in nonbalding DP cells, which displayed that the level of ß-catenin and LEF1 was significantly inhibited; however, the level of active TGF-ß1 and the phosphorylation of Smad2 and Akt were up-regulated. CONCLUSIONS: These data indicate that crosstalk between Wnt/ß-catenin and TGF-ß signaling pathways may exist as one of the important mechanisms contributing to AGA.

Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling.

Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant.

Astragaloside IV controls collagen reduction in photoaging skin by improving transforming growth factor-ß/Smad signaling suppression and inhibiting matrix metalloproteinase-1.

Exposure to ultraviolet (UV) light reduces levels of type I collagen in the dermis and results in human skin damage and premature skin aging (photoaging). This leads to a wrinkled appearance through the inhibition of transforming growth factor­ß (TGF­ß)/Smad signaling. UV irradiation increases type I collagen degradation through upregulating matrix metalloproteinase (MMP) expression. Astragaloside IV (AST) is one of the major active components extracted from Astragalus membranaceus. However, its multiple anti­photoaging effects remain to be elucidated. In the present study, the effects of AST against collagen reduction in UV­induced skin aging in human skin fibroblasts were investigated. The expression of type I procollagen (COL1), MMP­1, TGF­ßRⅡ and Smad7 were determined using reverse transcription­polymerase chain reaction, western blotting and ELISA, respectively. UV irradiation inhibits type I collagen production by suppressing the TGF­ß/Smad signaling pathway and increasing COL1 degradation by inducing MMP­1 expression. Transforming growth factor­ß type II protein and COL1 mRNA decreased but MMP­1 and Smad7 levels increased in the photoaging model group, which was reversed by topical application of AST. AST prevents collagen reduction from UV irradiation in photoaging skin by improving TGF­ß/Smad signaling suppression and inhibiting MMP­1, thus AST may be a potential agent against skin photoaging.

Effects of (-)-epigallocatechin-3-gallate on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibroblasts irradiated with ultraviolet A.

BACKGROUND: It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways. (-)-epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet-induced damage. In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro. METHODS: Transcription factor Jun protein levels were measured by Western blot. Matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA were studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in conjunction with computer-assisted image analysis. MMP-1 and TIMP-1 proteins were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: EGCG decreased transcription activity of Jun protein after induction by UVA. Both the mRNA and protein levels of MMP-1 were increased by UVA irradiation, while no significant changes were observed in TIMP-1 levels. The ratio of MMP-1 to TIMP-1 showed statistically significant differences compared with the control. EGCG decreased the ratio of MMP-1 to TIMP-1 by inhibiting UVA-induced MMP-1 expression (P < 0.05). CONCLUSION: EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP-1. The ratio of MMP-1 to TIMP-1, rather than the levels of MMP-1 or TIMP-1 alone, may play a significant role in human skin photodamage.

Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells.

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.

Perifosine sensitizes UVB-induced apoptosis in skin cells: new implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.

[Construction and immunogenicity evaluation of chimerical DNA vaccine of human papillomavirus type 11].

OBJECTIVE: To construct chimerical DNA vaccine plasmid of human papillomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. METHODS: Molecular cloning techniques were used to construct recombinant plasmid pcDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection.IL-2 and gamma-INF secreted by immunized spleens lymphocyte and HPV 11 L1 or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. RESULTS: The chimerical DNA plasmid of pcDNA3 L1-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and gamma-INF were detected in vaccinated mice. CONCLUSION: Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

[Physical and chemical characters of recombinant human nucleoside diphosphate kinase A].

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.