Staphylococcal Enterotoxin C2 Mutant-Induced Antitumor Immune Response Is Controlled by CDC42/MLC2-Mediated Tumor Cell Stiffness.

As a biological macromolecule, the superantigen staphylococcal enterotoxin C2 (SEC2) is one of the most potent known T-cell activators, and it induces massive cytotoxic granule production. With this property, SEC2 and its mutants are widely regarded as immunomodulating agents for cancer therapy. In a previous study, we constructed an MHC-II-independent mutant of SEC2, named ST-4, which exhibits enhanced immunocyte stimulation and antitumor activity. However, tumor cells have different degrees of sensitivity to SEC2/ST-4. The mechanisms of immune resistance to SEs in cancer cells have not been investigated. Herein, we show that ST-4 could activate more powerful human lymphocyte granule-based cytotoxicity than SEC2. The results of RNA-seq and atomic force microscopy (AFM) analysis showed that, compared with SKOV3 cells, the softer ES-2 cells could escape from SEC2/ST-4-induced cytotoxic T-cell-mediated apoptosis by regulating cell softness through the CDC42/MLC2 pathway. Conversely, after enhancing the stiffness of cancer cells by a nonmuscle myosin-II-specific inhibitor, SEC2/ST-4 exhibited a significant antitumor effect against ES-2 cells by promoting perforin-dependent apoptosis and the S-phase arrest. Taken together, these data suggest that cell stiffness could be a key factor of resistance to SEs in ovarian cancer, and our findings may provide new insight for SE-based tumor immunotherapy.

Whole-Genome Sequencing of a Chlorimuron-Ethyl-Degrading Strain: Chenggangzhangella methanolivorans CHL1 and Its Degrading Enzymes.

Chlorimuron-ethyl is a commonly used sulfonylurea herbicide, and its long-term residues cause serious environmental problems. Biodegradation of chlorimuron-ethyl is effective and feasible, and many degrading strains have been obtained, but still, the genes and enzymes involved in this degradation are often unclear. In this study, whole-genome sequencing was performed on chlorimuron-ethyl-degrading strain, Chenggangzhangella methanolivorans CHL1. The complete genome of strain CHL1 contains one circular chromosome of 5,542,510 bp and a G+C content of 68.17 mol%. Three genes, sulE, pnbA, and gst, were predicted to be involved in the degradation of chlorimuron-ethyl, and this was confirmed by gene knockout and gene complementation experiments. The three genes were cloned and expressed in Escherichia coli BL21 (DE3) to allow for the evaluation of the catalytic activities of the respective enzymes. The glutathione-S-transferase (GST) catalyzes the cleavage of the sulfonylurea bridge of chlorimuron-ethyl, and the esterases, PnbA and SulE, both de-esterify it. This study identifies three key functional genes of strain CHL1 that are involved in the degradation of chlorimuron-ethyl and also provides new approaches by which to construct engineered bacteria for the bioremediation of environments polluted with sulfonylurea herbicides. IMPORTANCE Chlorimuron-ethyl is a commonly used sulfonylurea herbicide, worldwide. However, its residues in soil and water have a potent toxicity toward sensitive crops and other organisms, such as microbes and aquatic algae, and this causes serious problems for the environment. Microbial degradation has been demonstrated to be a feasible and promising strategy by which to eliminate xenobiotics from the environment. Many chlorimuron-ethyl-degrading microorganisms have been reported, but few studies have investigated the genes and enzymes that are involved in the degradation. In this work, two esterase-encoding genes (sulE, pnbA) and a glutathione-S-transferase-encoding gene (gst) responsible for the detoxification of chlorimuron-ethyl by strain Chenggangzhangella methanolivorans CHL1 were identified, then cloned and expressed in Escherichia coli BL21 (DE3). These key chlorimuron-ethyl-degrading enzymes are candidates for the construction of engineered bacteria to degrade this pesticide and enrich the resources for bioremediating environments polluted with sulfonylurea herbicides.

Induction of CD4+ regulatory T cells by stimulation with Staphylococcal Enterotoxin C2 through different signaling pathways.

As a member of superantigens, Staphylococcal Enterotoxin C2 (SEC2) can potently activate T cells expressing specific Vß repertoires and has been applied in clinic for tumor immunotherapy in China for more than 20 years. However, excessive activation of T cells by over-stimulation with superantigen are always followed by eliciting regulatory T cells (Tregs) induction and functional immunosuppression, which brings uncertainties to SEC2 application in tumor immunotherapy. In this study, we found that SEC2 could induce CD4+CD25+Foxp3+ Tregs from the murine splenocytes in dose and time related manners. The induced Tregs with high expression of GITR and CTLA-4 and low expression of CD127 were TCR Vß8.2-specific and have character of IL-10 production in a SEC2 dose-depended manner. Importantly, SEC2-induced CD4+ Tregs showed the potent capacity of suppressing proliferation of intact murine splenocytes response to SEC2. Furthermore, by using specific inhibitors or neutralizing antibody, we proved that the signaling pathways of TCR-NFAT/AP-1, IL-2-STAT5, and TGF-ß-Smad3 play crucial roles in Tregs induction by SEC2. These findings will help us better understand the balance of immune stimulation and immunosuppression mediated by SEC2 and provide valuable guidance for SEC2 application in antitumor immunology.

TRAIL produced by SAM-1-activated CD4+ and CD8+ subgroup T cells induces apoptosis in human tumor cells through upregulation of death receptors.

Bacterial superantigens potently activate conventional T-cells to induce massive cytokine production and mediate tumor cell death. To engineer superantigens for immunotherapy against tumors in clinic, we previously generated SAM-1, a staphylococcal enterotoxins C2 (SEC2) mutant, that exhibited significantly reduced toxicity but maintained the superantigen activity in animal models. This present study aimed to investigate whether SAM-1 activates T cells and induces apoptosis in human tumor cells. We found that SAM-1 induced the maturation of dendritic cells (DCs) with upregulating expression of the surface markers CD80, CD86 and HLA-DR, which secreted high levels of IL-12p70 by activating TLR2-NF-κB signaling pathways. SAM-1 could activate human CD4+ subgroup T cells and CD8+ subgroup T cells in the presence of mature dendritic cells (DCs), leading to the productions of cytokines TRAIL, IL-2, IFN-γ and TNF-α. We observed that TRAIL mediated the apoptosis and S-phase and G2/M-phase arrest in HGC-27 tumor cells via binding to upregulated death receptors DR4 and DR5. Using shRNA knockdown in HGC-27 cells or constitutive overexpression in ES2 cells for DR4 and DR5, we demonstrated the vital requirement of DR4 and DR5 in apoptosis of tumor cells in response to TRAIL secreted from SAM-1-activated T cells. Collectively, our results will facilitate better understanding of SAM-1-based immunotherapies for cancer.

Staphylococcal Enterotoxin C2 Mutant-Directed Fatty Acid and Mitochondrial Energy Metabolic Programs Regulate CD8+ T Cell Activation.

CD8+ T cells can switch between fatty acid catabolism and mitochondrial energy metabolism to sustain expansion and their cytotoxic functions. ST-4 is a TCR-enhanced mutant derived from superantigen staphylococcal enterotoxin C2 (SEC2), which can hyperactivate CD4+ T cells without MHC class II molecules. However, whether ST-4/SEC2 can enhance metabolic reprogramming in CD8+ T cells remains poorly understood. In this study, we found that ST-4, but not SEC2, could induce proliferation of purified CD8+ T cell from BALB/c mice in Vß8.2- and -8.3-specific manners. Results of gas chromatography-mass spectroscopy analysis showed that fatty acid contents in CD8+ T cells were increased after ST-4 stimulation. Flow cytometry and Seahorse analyses showed that ST-4 significantly promoted mitochondrial energy metabolism in CD8+ T cells. We also observed significantly upregulated levels of gene transcripts for fatty acid uptake and synthesis, and significantly increased protein expression levels of fatty acid and mitochondrial metabolic markers of mTOR/PPARγ/SREBP1 and p38-MAPK signaling pathways in ST-4-activated CD8+ T cells. However, blocking mTOR, PPARγ, SREBP1, or p38-MAPK signals with specific inhibitors could significantly relieve the enhanced fatty acid catabolism and mitochondrial capacity induced by ST-4. In addition, blocking these signals inhibited ST-4-stimulated CD8+ T cell proliferation and effector functions. Taken together, our findings demonstrate that ST-4 enhanced fatty acid and mitochondria metabolic reprogramming through mTOR/PPARγ/SREBP and p38-MAPK signaling pathways, which may be important regulatory mechanisms of CD8+ T cell activation. Understanding the effects of ST-4-induced regulatory metabolic networks on CD8+ T cells provide important mechanistic insights to superantigen-based tumor therapy.

An iRGD peptide fused superantigen mutant induced tumor-targeting and T lymphocyte infiltrating in cancer immunotherapy.

Solid tumors are intrinsically resistant to immunotherapy because of the major challenges including the immunosuppression and poor penetration of drugs and lymphocytes into solid tumors due to the complicated tumor microenvironment (TME). Our previous study has created a novel superantigen mutant ST-4 to efficiently active the T lymphocytes and alleviate immune suppression. In the present study, to accumulate ST-4 into the TME, we constructed a recombinant protein, ST-4-iRGD, by fusing ST-4 to a tumor-homing peptide, iRGD. We hypothesized that ST-4-iRGD could internalize into the TME through iRGD-mediated tumor targeting and tumor tissue penetrating to activate the regional immunoreaction. The results of in vitro studies showed that ST-4-iRGD achieved improved tumor targeting and cytotoxicity in mouse B16F10 melanoma cells. The iRGD-mediated tumor tissue penetration was further confirmed by imaging and immunofluorescence studies in vivo, wherein higher distribution of ST-4-iRGD was observed in the mouse 4T1 breast tumor model. Moreover, ST-4-iRGD exhibited enhanced anti-solid tumor characteristics and induced improved lymphocyte infiltration in the B16F10 and 4T1 models. In conclusion, using iRGD to facilitate better dissemination of the therapeutic agent ST-4 throughout a solid tumor mass is feasible, and ST-4-iRGD may be a potential candidate for efficient cancer immunotherapy in the future.

Internalization and toxicity: A preliminary study of effects of nanoplastic particles on human lung epithelial cell.

As a kind of newly emerging pollutant, nanoplastics are easily to be ingested by organisms, and cause severe damage to biological functions because of their small size, high specific surface area, and strong biological penetration. Recently, there are increasing reports of numerous airborne microplastics, including polystyrene (PS), being detected in atmospheric samples, which implies a potential risk to the human respiratory system. In this work, we evaluated the effects of polystyrene nanoparticles of two different sizes (PS-NP25: 25 nm diameter and PS-NP70: 70 nm diameter) on the human alveolar epithelial A549 cell line including internalization, cell viability, cell cycle, apoptosis, and associated gene transcription and protein expression. Results showed that PS-NP25 was internalized more rapidly and efficiently into the cytoplasm of A549 than PS-NP70. PS-NPs significantly affected the cell viability, caused cell cycle S phrase arrest, activated inflammatory gene transcription, and changed the expression of proteins associated with cell cycle and pro-apoptosis. PS-NPs induced significant up-regulation of pro-inflammatory cytokines such as IL-8, NF-κB, and TNF-α, as well as pro-apoptotic proteins such as DR5, caspase-3, caspase-8, caspase-9, and cytochrome c, which revealed that PS-NPs triggered a TNF-α-associated apoptosis pathway. This study suggests that exposure duration, diameter, and concentration are the key factors for evaluating the toxicological effects of PS-NPs on alveolar epithelial cells. More attention must be focused on the risk of nanoplastic-related air pollution and the environmental toxicological effects of nanoplastics on humans and other terrestrial mammals.

Enhanced interaction between SEC2 mutant and TCR Vß induces MHC II-independent activation of T cells via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways.

SEC2, a major histocompatibility complex class II (MHC II)-dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) Vßs to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR Vßs, ST-4, which could intensify NF-κB signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4+ T-cell proliferation in a Vß8.2- and Vß8.3-specific manner in the absence of MHC II+ antigen-presenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase Cθ (PKCθ), inhibitor of κB (IκB), α and ß IκB kinase (IKKα/ß), IκBα, and NF-κB in mouse splenocytes, only ST-4 could activate CD4+ T cells in the absence of MHC II+ APCs through the PKCθ/NF-κB signaling pathway. The PKCθ inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II+ APCs. Further, SEC2/ST-4-induced changes in PKCθ/NF-κB signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKCθ. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4+ T-cell proliferation via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKCθ/NF-κB and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.

Staphylococcal enterotoxin C2 stimulated the maturation of bone marrow derived dendritic cells via TLR-NFκB signaling pathway.

As a kind of superantigen, staphylococcal enterotoxin C2 (SEC2) is well known as a powerful immunomodulator. However, most previous studies about SEC2 focus on its T cell activating characters. But the direct effect of SEC2 on antigen-presenting cells (APCs) which are important for the T cell activation is not clearly. In this study, we investigated the effect of SEC2 on murine bone marrow-derived dendritic cells (BMDCs) which are known as the specialized professional APCs. Contrary to its effects on T cells, SEC2 could not induce proliferation or cytotoxicity to BMDCs even in high concentrations. While SEC2 could promote the mature of BMDCs with increased expression of co-stimulatory molecules on cell membrane such as CD80, CD86, and MHC II. The production of pro-inflammatory cytokines such as TNF-α, IFN-γ and IL-6 were also increased in BMDCs treated with SEC2. We also found that SEC2 enhanced the genes expression of pattern recognition receptors including toll-like receptors 2 (TLR2) and TLR4 in BMDCs, and up-regulated the key signal molecule MyD88 in both mRNA and protein levels. In addition, SEC2 also caused IκBα degradating and NFκB p65 translocating from the cytoplasm to the nucleus in BMDCs. The siRNAs for both TLR2 and TLR4, as well as NFκB specific inhibitor BAY 11-7085 could inhibit the co-stimulatory molecules expression and pro-inflammatory cytokines releasing induced by SEC2. Moreover, TLR2/4 specific siRNAs inhibited p65 and MyD88 upregulation induced by SEC2. In summary, all our results indicated that SEC2 could stimulate BMDCs maturation through TLR-NFκB signaling pathways.

[Construction of HER2-specific CAR-T cells and in vitro analysis of their activity to suppress tumor cell growth].

CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2 (HER2) was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was (58.47±1.72)%, significantly higher than that on the mock-treated control group (P<0.05). The inhibitory rate on HER2-negative K562 cell lines was (11.74±2.37)%, which was not significantly different from that on the control group (P>0.05). Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to (30.41±7.59)%, which was higher than that on HER2-negative K562 (P<0.05). Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.

Superantigen staphylococcal enterotoxin C1 inhibits the growth of bladder cancer.

Superantigens can induce cell-mediated cytotoxicity preferentially against MHC II-positive target cells with large amounts of inflammatory cytokines releasing. In this study, superantigen staphylococcal enterotoxin C (SEC) 1 was investigated to evaluate its potential in bladder cancer immunotherapy in vitro and in vivo. Our results revealed that SEC1 could stimulate the proliferation of human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner, accompanied with the release of interleukin-2, interferon-γ, and tumor necrosis factor-α, and increased the population of CD4+ T cells and CD8+ T cells. PBMCs stimulated by SEC1 could initiate significant cytotoxicity towards human bladder cancer cells in vitro. The results of in vivo antitumor experiment indicated that SEC1 could decrease the rate of tumor formation and prolong the survival time of tumor-bearing mice. Our study demonstrated that SEC1 inhibited the growth of bladder cancer. And it is also suggested that SEC1 may become a candidate for bladder cancer immunotherapy.

Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1-6 cells.

Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vß 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future.

TNF-α produced by SEC2 mutant (SAM-3)-activated human T cells induces apoptosis of HepG2 cells.

Staphylococcal enterotoxins C2 (SEC2) is a classical model of superantigens (SAg), which has the powerful ability to activate T cells as well as induce massive cytokine production. This property makes SEC2 and its mutants well concerned as a potential new immune-regulatory agent for cancer therapy. We previously constructed a SEC2 mutant named SAM-3, which had prominently antitumor activity in BALB/c mice model. But, the underlying molecular mechanism for stimulation of human peripheral blood mononuclear cells (PBMCs) and antitumor effect on human tumor cells induced by SAM-3 is not clear. Here, we showed that SAM-3 could activate human TCR Vß 12, 13A, 14, 15, 17, and 20 CD8(+) subgroup T cells, which secreted the cytokines IL-2, IFN-γ, and TNF-α, and exhibit stimulation activity in a dose-dependent manner. TNF-α secreted from activated T cells could induce apoptosis and G1-phase arrest and lead to the antitumor effect in HepG2 cells. Meanwhile, SAM-3 upregulated the expression of tumor necrosis factor receptor 1 (TNFR1) mRNA and activity of caspase-3 and caspase-8. We also found that the antitumor activity and activity of caspase-3 and caspase-8 were decreased when the neutralizing TNF-α monoclonal antibody presented. These data suggest that TNF-α secreted by SAM-3-activated T cells is an important factor in inducing apoptosis in HepG2 cells.

[Enhanced SEC2 mutants and their superantigen activities].

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.

SEC2-induced superantigen and antitumor activity is regulated through calcineurin.

Once the TCR-SAg-MHC II ternary complex is established, it triggers a variety of intracellular signal transduction pathways, which provoke extreme responses in the immune system. However, the signaling events that involved in SAg-induced immune activation are not well understood. In this study, we demonstrated that the Ca(2+)/calcineurin (CaN)/nuclear factor of activated T cells (NFAT) signaling pathway was involved in SEC2-induced immune activation, and selective blockade of CaN by its inhibitor cyclosporine A (CsA) can completely inhibited the SEC2-induced T-cell stimulating potency. In addition, we selected an engineered SEC2 mutant named SAM-1 based on a series of biological activity tests, and our further studies on it not only confirmed that the CaN activity and gene transcription of its key substrates were proportional to the SEC2/SAM-1-induced T-cell stimulating potency, but also suggested that intensified Ca(2+)/CaN/NFAT signaling transduction induced by SAM-1 resulted in enhanced T-cell stimulating potency, production of cytokines and cytotoxicity, which finally elicit the improved antitumor activity of SAM-1 in vivo.

T-cell proliferation and antitumour activities of a truncated mutant of staphylococcal enterotoxin C2 with decreased cytokine secretion.

As a superantigen, staphylococcal enterotoxin C2 (SEC2) has commonly been used as an antitumour immunotherapy agent in China. However, the clinical application of SEC2 has been hampered by its pyrogenic toxicity and the presence of neutralizing antibody in patients. Thus, an improvement in its superantigen-based immunotherapy is highly needed. In this study, a truncated SEC2 mutant, SEC(14-128), was constructed without the N-terminal 13 and C-terminal 111 aa. This mutant retained T-cell proliferation and antitumour activities in in vitro experiments. However, it induced a significantly decreased release of the main inflammatory cytokines inerleukin-2 and gamma interferon. Moreover, SEC(14-128) exhibited reduced toxicity and affinity to anti-SEC2 IgG compared with native SEC2. Based on the considerable antitumour activity and low toxicity, it is proposed that the mutant SEC(14-128) could be a potential candidate for cancer treatment.

iASPP is important for bladder cancer cell proliferation.

Inhibitor of apoptosis stimulatory protein phosphatase (iASPP) is a key inhibitor of p53 conserved from worm to human and is associated with cell proliferation and carcinogenesis in a variety of human cancers. Because iASPP is important for tumor cell apoptosis, it is a potential target for cancer gene therapy. However, it is still not clear whether iASPP is relevant to p53-deficient human bladder cancer. In the present study, iASPP was knocked down in bladder carcinoma 5637 and T24 cells (p53 defective) by lentiviral-mediated interfering short hairpin RNAs (siRNAs). MTT assay, BrdU incorporation assay, and colony formation assay were performed to investigate the role of iASPP on cell proliferation. It was suggested that iASPP knockdown led to cell growth deceleration and slow colony formation. A positive relationship between expression of iASPP and bladder cancer proliferation was found. The expression of iASPP may be critical for proliferation of bladder cancer cells. Our study indicates iASPP could be an important target for therapy in bladder cancer.

An engineered superantigen SEC2 exhibits promising antitumor activity and low toxicity.

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.

Biological characterization of the zinc site coordinating histidine residues of staphylococcal enterotoxin C2.

The bacterial toxin staphylococcal enterotoxin C2 (SEC2) can cause staphylococcal toxic shock syndrome and food poisoning. Although the previously determined crystal structure of SEC2 revealed that some histidine residues (His47, His118 and His122) contribute to the binding of zinc ions, little is known about their biological roles in SEC2. This prompted us to investigate the role of the zinc site coordinating histidine residues in the biological activities of SEC2. The mutants with substitutions at positions 118 and 122 all retained T-cell stimulatory activity, whereas the histidine mutants at position 47 were defective in the ability to stimulate T-cell proliferation. Further toxicity assays in vivo indicated that mutants SEC2-H118A and SEC2-H122A were defective in emetic and febrile activities. However, mutant SEC2-H47A could cause significant emetic and febrile responses in comparison with the other two histidine mutants. These findings suggested that the zinc-coordinating histidine residues play significant roles in superantigen and toxic activities of SEC2 and further implied that superantigen and febrile activities could be separable in staphylococcal enterotoxins. The results also show that it should be possible to design new SEC2 immunotherapeutic agents that have superantigen activity and low toxicity.

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis.

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vbeta domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immunotherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.