Manipulating TGF-ß signaling to optimize immunotherapy for cervical cancer.

Cervical cancer is a serious threat to women's health globally. Therefore, identifying key molecules associated with cervical cancer progression is essential for drug development, disease monitoring, and precision therapy. Recently, TGF-ß (transforming growth factor-beta) has been identified as a promising target for cervical cancer treatment. For advanced cervical cancer, TGF-ß participates in tumor development by improving metastasis, stemness, drug resistance, and immune evasion. Accumulating evidence demonstrates that TGF-ß blockade effectively improves the therapeutic effects, especially immunotherapy. Currently, agents targeting TGF-ß and immune checkpoints such as PD-L1 have been developed and tested in clinical studies. These bispecific antibodies might have the potential as therapeutic agents for cervical cancer treatment in the future.

Predictive value of the systemic immune-inflammation index for the efficacy of neoadjuvant chemotherapy and prognosis in patients with stage III ovarian cancer-a retrospective cohort study.

Background: The systemic immune-inflammation index (SII) has been proven to be related to the prognoses of multiple malignant tumors. However, there are still few studies on the relationship between the SII and the effect of neoadjuvant chemotherapy in patients with ovarian cancer. It is of great significance to predict the efficacy of neoadjuvant chemotherapy in patients with ovarian cancer. Our study was aimed at determining the predictive value of the SII for the efficacy of neoadjuvant chemotherapy and prognosis in patients with stage III ovarian cancer. Methods: A total of 102 patients with stage III ovarian cancer treated in Tongji Hospital of Tongji University from January 2017 to January 2019 were retrospectively collected. According to the level of the SII before neoadjuvant chemotherapy, patients were divided into the high SII group and low SII group. We compared the effect of neoadjuvant chemotherapy between the 2 groups, and observed the progression-free survival and mortality of patients in the 2 groups after 3 years follow-up. Results: Compared with patients in the low SII group, the complete response rate of patients in the high SII group decreased significantly after neoadjuvant chemotherapy (13.73% vs. 45.10%, P=0.001), and the progressive disease rate increased (19.61% vs. 1.96%, P=0.011). The SII had certain value in predicting the inefficacy of neoadjuvant chemotherapy in patients with stage III ovarian cancer, and the area under the curve was 0.655 (95% CI: 0.548-0.762, P=0.007). The progression-free survival of patients in the high SII group was shorter than that of patients in the low SII group (P<0.001), and the overall survival rate of patients in the high SII group was lower (47.06% vs. 70.59%, P=0.016). The SII had predicting value for the postoperative death of ovarian cancer patients after neoadjuvant chemotherapy, and the area under the curve was 0.646 (95% CI: 0.537-0.756, P=0.012). Multivariate regression analysis showed that higher SII was a risk factor for death in ovarian cancer patients after neoadjuvant chemotherapy (OR: 2.700, P=0.017). Conclusions: A high SII was a predictor of inefficacy of neoadjuvant chemotherapy in patients with stage III ovarian cancer and was related to poor prognosis.

YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation.

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells.

Women with triple negative breast cancer (TNBC) have poor prognosis compared to other breast cancer subtypes. There were several reports indicating racial disparity in breast cancer outcomes between African American (AA) and European American (EA) women. For example, the mortality rates of AA breast cancer patients were three times higher than of EA patients, even though, the incidence is lower in AA women. Our in vitro studies indicate that cancer stem-like cells (CSCs) derived from AA TNBC cell lines have significantly higher self-renewal potential (mammosphere formation) than CSCs derived from EA cell lines. TNBC tumors express high levels of Myc compared to luminal A or HER2 expressing breast cancers. We studied the effects of c-Myc overexpression on CSCs and chemotherapy in AA, and EA derived TNBC cell line(s). Overexpression of c-Myc in AA derived MDA-MB-468 (Myc/MDA-468) cells resulted in a significant increase in CSCs and with minimal changes in epithelial-to-mesenchymal transition (EMT) compared to the control group. In contrast, overexpression of c-Myc in EA derived MDA-MB-231(Myc/MDA-231) cells led to increased epithelial-to-mesenchymal transition (EMT), with a minimal increase in CSCs compared to the control group. Myc/MDA-468 cells were resistant to standard chemotherapeutic treatments such as iniparib (PARP inhibitor) plus cisplatin, / iniparib, cisplatin, paclitaxel and docetaxel. However, Myc/MDA-231 cells, which showed EMT changes responded to iniparib with cisplatin, but were resistant to other drugs, such as iniparib, cisplatin, paclitaxel and docetaxel. Collectively, our results indicate that intrinsic differences in the tumor biology may contribute to the breast cancer disparities.

Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4.

BACKGROUND: For breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors, treatment with tamoxifen is the gold standard. A significant number of patients, however, develop resistance to tamoxifen, and management of such tamoxifen-resistant patients is a major clinical challenge. With an eye to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed that tamoxifen-resistant cells derived from ER-positive MCF-7 cells (MCF7TR) exhibit an increased expression of microRNA-10b (miR-10b). A role of miR-10b in drug-resistance of breast cancer cells has never been investigated, although its is very well known to influence invasion and metastasis. METHODS: To dileneate a role of miR-10b in tamoxifen-resistance, we over-expressed miR-10b in MCF-7 cells and down-regulated its levels in MCF7TR cells. The mechanistic role of HDAC4 in miR-10b-mediated tamoxifen resistance was studied using HDAC4 cDNA and HDAC4-specific siRNA in appropriate models. RESULTS: Over-expression of miR-10b in ER-positive MCF-7 and T47D cells led to increased resistance to tamoxifen and an attenuation of tamoxifen-mediated inhibition of migration, whereas down-regulation of miR-10b in MCF7TR cells resulted in increased sensitivity to tamoxifen. Luciferase assays identified HDAC4 as a direct target of miR-10b. In MCF7TR cells, we observed down-regulation of HDAC4 by miR-10b. HDAC4-specific siRNA-mediated inactivation of HDAC4 in MCF-7 cells led to acquisition of tamoxifen resistance, and, moreover, reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen-resistance. CONCLUSIONS: We propose miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance which can potentially be expolited as a novel targeted therapeutic approach for the clinical management of tamoxifen-resistant breast cancers.

Anti-estrogen­resistant breast cancer cells are sensitive to cisplatin plus TRAIL treatment.

Breast cancer patients who are positive for estrogen receptor (ER) are usually treated with anti-estrogen drugs, such as tamoxifen (Tam). However, a great majority of such patients eventually develop resistance to Tam. In this study, MCF-7 cells (with de novo and/or acquired resistance to Tam) as well as T47D cells (acquired resistance to Tam) models were used to investigate the effect of treatment with cisplatin plus tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The results in the two cell types treated with cisplatin plus TRAIL showed significantly increased cell death compared to that in the untreated control cells. A similar treatment had a minimal effect on normal breast cells, the increased cell death appeared to be caused by the activation of caspases and, inhibition of the activity of caspases (using relatively specific inhibitors) reduced the cell death caused by cisplatin plus TRAIL treatment. Taken together, the results suggested that cisplatin plus TRAIL treatment has the potential to provide a novel treatment strategy to improve the treatment outcome in anti-estrogen­resistant breast cancer patients.

Tumor-initiating cells and FZD8 play a major role in drug resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women, whereas the majority who had residual tumor had a relatively poor outcome. To identify the mechanism by which residual cancer cells survive chemotherapy, we initially conducted gene expression profiling using the CRL2335 TNBC cell line derived from a squamous breast carcinoma before and after treatment with cisplatin plus TRAIL. We found a significant increase in the expression of FZD8, one of Wnt receptors, and its downstream targets LEF1 and TCF in residual CRL2335 tumor cells after treatment with cisplatin plus TRAIL. Increased FZD8 levels were further confirmed in other TNBC cell lines. Inhibition of FZD8 by siRNA in CRL2335 cells in the presence of cisplatin plus TRAIL reduced ß-catenin and survivin levels and increased apoptosis compared with scrambled siRNA-treated cells. In vivo data show that cisplatin plus TRAIL treatment significantly reduces tumor volume in NOD/SCID mice. However, we found that cisplatin plus TRAIL treatment predominantly eliminated non-tumor-initiating cells, as shown by whole-body fluorescent imaging of mice injected with mammosphere-forming CRL2335 cells stably transfected with DsRed. This led to TIC enrichment in residual tumors, as confirmed by immunostaining for TIC markers. Moreover, an increase in FZD8 expression was observed in residual tumors treated with cisplatin and TRAIL. Taken together, our findings suggest that FZD8-mediated Wnt signaling may play a major role in mediating resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBCs.

Cisplatin and TRAIL enhance breast cancer stem cell death.

Triple negative breast cancer (TNBC) has increased recurrence and poor survival, despite a high response rate to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicate that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, ß-catenin, phospho ß-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL-treated cells. Taken together the data suggest that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Enhanced anticancer effect of the combination of cisplatin and TRAIL in triple-negative breast tumor cells.

Women with triple-negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors, such as ER, PR, and HER-2 amplification. Conventional chemo- and/ or radiation therapy also seems to have limited efficacy in TNBC patients. We studied the effects of cisplatin plus TRAIL on 1 normal and 2 TNBC cells in vitro. The in vitro studies indicate that cisplatin plus TRAIL significantly enhanced cell death in TNBC cell lines CRL2335 and MDA-MB-468 by approximately 60%-70% compared with approximately 10%-15% in CRL8799 normal breast cell line. Treatment with cisplatin/TRAIL also inhibited the expression of EGFR, p63, survivin, Bcl-2, and Bcl-xL in TNBC cells. Specific inhibition of EGFR and/or p63 protein in TNBC cells by small interfering RNA (siRNA) does not increase TRAIL-induced apoptosis. However, inhibition of survivin by siRNA enhances TRAIL-induced apoptosis. These observations suggested the possibility that survivin played an important role in cisplatin plus TRAIL-induced apoptosis in TNBC cells. In vivo experiments, treatment of mice with cisplatin plus TRAIL resulted in a significant inhibition of CRL2335 xenograft tumors compared with untreated control tumors. Taken together the data suggest that cisplatin plus TRAIL treatment have the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients.

Maspin reprograms the gene expression profile of prostate carcinoma cells for differentiation.

Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFß signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.

Protein kinase Cδ and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL-induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL-induced apoptosis was significantly inhibited in PKCδ overexpressing MCF-7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase-8 activation, a first step in TRAIL-induced apoptosis, thus preventing TRAIL-induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase-8 activation leading to TRAIL-induced apoptosis. To determine if caspase-3-induced PKCδ cleavage reverses its inhibition on caspase-8, we developed stable cell lines that either expresses wild-type PKCδ (MCF-7/cas-3/PKCδ) or caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδ mut) utilizing MCF-7 cells expressing caspase-3. Cells that overexpress caspase-3 cleavage-resistant PKCδ mutant (MCF-7/cas-3/PKCδmut) significantly inhibited TRAIL-induced apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. In MCF-7/cas-3/PKCδmut cells, TRAIL-induced caspase-8 activation was blocked leading to inhibition of apoptosis when compared to wild-type PKCδ (MCF-7/cas-3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase-8 activation leading to inhibition of TRAIL-induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase-3 sensitizes cells to TRAIL-induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy.

Maspin augments proteasome inhibitor-induced apoptosis in prostate cancer cells.

Proteasome inhibitors are known to induce apoptosis in a variety of cancer cells. On the other hand, maspin, a non-inhibitory serine protease inhibitor, is shown to sensitize cancer cells to therapeutic agents that induce apoptosis. We examined the consequence of maspin expression in prostate cancer cells targeted for treatment with various proteasome inhibitors. We observed that proteasome inhibitors induced apoptosis more effectively in maspin transfected human prostate cancer DU145 cells than in control cells. Interestingly, increased apoptosis in these cells was associated with a significant induction of maspin expression. MG-132, a proteasome inhibitor, induced endogenous and ectopic [cytomegalovirus promoter (CMV)-driven] maspin expression, and maspin siRNA attenuated MG-132-induced apoptosis. Proteasome inhibitor-induced maspin expression was inhibited by actinomycin D (Act D) and cyclohexamide (CHX), and by the inhibitors of p38MAPK, but not ERK1/2 or NF-kappaB. Electrophoretic mobility-shift assay (EMSA) and promoter-reporter activity analyses suggested that p38MAPK activated transcription factor AP-1 is responsible for proteasome inhibitor-induced maspin expression. Taken together, these observations demonstrate that proteasome inhibitors induce maspin expression by activating p38MAPK pathway, and that maspin thus expressed, in turn, augments proteasome inhibitor-induced apoptosis in prostate cancer cells. Our results suggest that gene therapy involving ectopic maspin expression may dramatically improve the efficacy of proteasome inhibitors for the treatment of prostate cancer.

Endogenous inhibition of histone deacetylase 1 by tumor-suppressive maspin.

Maspin, a noninhibitory serine protease inhibitor, exerts multifaceted tumor-suppressive effects. Maspin expression is associated with better differentiated phenotypes, better cancer prognosis, and better drug sensitivity. Consistently, maspin also correlates with increased expression of Bax and p21WAF1/CIP1. Interestingly, histone deacetylase 1 (HDAC1), a major HDAC responsible for histone deacetylation, was shown to interact with maspin in a yeast two-hybrid screening. In this study, we confirmed the maspin/HDAC1 interaction in human prostate tissues, in prostate cancer cell lines, and with purified maspin. We produced several lines of evidence that support an inhibitory effect of maspin on HDAC1 through direct molecular interaction, which was detected in both the nucleus and the cytoplasm. Both endogenously expressed maspin and purified maspin inhibited HDAC1. In contrast, small interfering RNA (siRNA) silencing of maspin in PC3 cells increased HDAC activity. Accordingly, maspin-transfected DU145 cells exhibited increased expression of HDAC1 target genes Bax, cytokeratin 18 (CK18), and p21(WAF1/CIP1), whereas maspin siRNA decreased CK18 expression in PC3 cells. The maspin effect on HDAC1 correlated with an increased sensitivity to cytotoxic HDAC inhibitor M344. Interestingly, glutathione S-transferase (GST, another maspin partner) was detected in the maspin/HDAC1 complex. Furthermore, a COOH-terminally truncated maspin mutant, which bound to HDAC1 but not GST, did not increase histone acetylation. Although HDACs, especially the highly expressed HDAC1, are promising therapeutic targets in cancer intervention, our data raise a novel hypothesis that the endogenous inhibitory effect of maspin on HDAC1 is coupled with glutathione-based protein modification, and provide new leads toward future developments of specific HDAC1-targeting strategies.

Maspin retards cell detachment via a novel interaction with the urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor system.

It is well documented that tumor suppressive maspin inhibits tumor cell invasion and extracellular matrix remodeling. Maspin is a cytosolic, cell surface-associated, and secreted protein in the serine protease inhibitor superfamily. Although several molecules have been identified as candidate intracellular maspin targets, the extracellular maspin target(s) remains elusive. Although maspin does not directly inhibit urokinase-type plasminogen activator (uPA) activity, we have shown evidence that maspin may block the pericellular proteolysis mediated by cell surface-associated uPA. In the current study, maspin significantly inhibited the Ca2+ reduction-induced detachment of DU145 cells. This maspin effect was associated with increased and sustained levels of mature focal adhesion contacts (FAC). We noted that maspin (a) colocalized with uPA and uPA receptor (uPAR), (b) enhanced the interaction between uPAR and low-density lipoprotein receptor related protein, and (c) induced rapid internalization of uPA and uPAR. The maspin effects on surface-associated uPA and uPAR required the interaction between uPA and uPAR. Further biochemical and biophysical analyses revealed that maspin specifically bound to pro-uPA with a deduced K(d) of 270 nmol/L and inhibited the plasmin-mediated pro-uPA cleavage. Interestingly, substitution of maspin p1' site Arg340 in the reactive site loop (RSL) with alanine not only abolished the binding to pro-uPA but also diminished the maspin effects on pro-uPA cleavage and cell detachment. These data show an important role of maspin RSL in regulating the uPA/uPAR-dependent cell detachment. Together, our data led to a new hypothesis that maspin may stabilize mature FACs by quenching localized uPA/uPAR complex before uPA activation.

Tumor suppressive maspin and epithelial homeostasis.

Maspin is a 42-kDa novel serine protease inhibitor (serpin) with multifaceted tumor suppressive activities. To date, the consensus that maspin expression predicts a better prognosis still largely holds for breast, prostate, colon, and oral squamous cancers. Interestingly, however, more detailed analyses revealed a biphasic expression pattern of maspin in early steps of tumorigenicity and re-expression of maspin in dormant cancer metastatic revertants. These data suggest a sensitivity of maspin expression to changes of epithelial microenvironments, and a role of maspin in epithelial homeostasis. Experimental evidence consistently showed that maspin suppresses tumor growth, invasion and metastasis, induces tumor redifferentiation, and enhances tumor cell sensitivity to apoptosis. Maspin protein isolated from biological sources is a monomer, which is present as a secreted, a cytoplasmic, a nuclear, as well as a cell surface-associated protein. Nuclear maspin is associated with better prognoses of cancer. It is further noted that extracellular maspin is sufficient to block tumor induced extracellular matrix degradation, tumor cell motility and invasion, whereas intracellular maspin is responsible for the increased cellular sensitivity to apoptosis. Despite these exciting developments, the mechanistic studies of maspin have proven challenging primarily due to the lack of a prototype molecular model. Although the maspin sequence has overall homologies with other members in the serpin superfamily, it does not behave like a typical serpin, that is, non-inhibitory toward active serine proteases in solution. This novel feature is in line with the X-ray crystallographic evidence. Several recent studies dedicated to finding the maspin partners support a paradigm shift. The current review is intended to summarize these recent findings and discuss a new perspective of maspin in epithelial homeostasis.

Tumor-suppressive maspin regulates cell response to oxidative stress by direct interaction with glutathione S-transferase.

Maspin, a novel serine protease inhibitor, suppresses tumor progression in several cancer models, including an in vivo model for prostate cancer bone metastasis. However, the molecular mechanism of maspin remains illusive, primarily because its molecular targets are unknown. To this end, we used a full-length maspin cDNA bait to screen against both a primary prostate tumor cDNA prey library and a HeLa cDNA prey library by the yeast two-hybrid method. We found that heat shock protein 90, glutathione S-transferase (GST), and heat shock protein 70 interacted with maspin with the highest frequencies. We confirmed the maspin/GST interaction using purified proteins, human epithelial cell lines, and human prostate tissues. A maspin variant that has a point mutation of Arg(340) to Ala (Mas(R340A)) showed a significantly decreased affinity for GST. Although purified maspin had no effect on the activity of purified GST in vitro, intracellular interaction between endogenous maspin and GST correlated with an elevated total GST activity in both MDA-MB-435- and DU145-derived stably transfected cells. Consistently, tumor cells treated with purified wild type maspin, but not Mas(R340A), enhanced cellular GST activity. Maspin expression in cancer cell lines also correlated with decreased basal levels of reactive oxygen species (ROS). Furthermore, H(2)O(2) treatment not only induced GST expression but also increased intracellular maspin/GST interaction, which was inversely correlated with the level of ROS generation. Conversely, maspin knockdown by small interfering RNA increased the basal, as well as H(2)O(2)-induced, ROS generation. Furthermore, the maspin effect on ROS generation was completely abolished by a GST inhibitor, indicating an essential role of GST in maspin-mediated cellular response to oxidative stress. Consistently, oxidative stress-induced vascular endothelial growth factor A expression was significantly inhibited in maspin-expressing cells. Together, our data suggest a new mechanism by which maspin, through its direct interaction with GST, may inhibit oxidative stress-induced ROS generation and vascular endothelial growth factor A induction, thus preventing further adverse effects on tumor genetics and stromal reactivity.

Human IgM monoclonal antibodies reactive with HIV-1-infected cells generated using a trans-chromosome mouse.

The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.

Bax mediates the apoptosis-sensitizing effect of maspin.

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.