Methylseleninic acid enhances paclitaxel efficacy for the treatment of triple-negative breast cancer

A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.     

A prostate cancer vaccine comprising whole cells secreting IL-7, effective against subcutaneous challenge,requires local GM-CSF for intra-prostatic efficacy

A panel of cytokine-secreting RM-9 prostate cancer cells were tested as whole cell vaccines to determine their capacity to evoke an anti-prostate cancer immune response. In our model, vaccines secreting mGM-CSF or mIL-7 resulted in the highest increase in circulating T lymphocytes after vaccination, prolonged survival and, in a proportion of animals, tumor-free survival. Anti-tumor effects were more evident after a subcutaneous RM-9 challenge than after an intraprostatic challenge. However, when the RM-9/mGM-CSF cell line was used as intraprostatic tumor challenge, protection after RM-9/mIL-7 vaccination was restored.     

Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES

Background

Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation.

Method

The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model.

Results

Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells.

Conclusion

Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.     

Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: role of IDO

We report that administration of celecoxib, a specific cyclooxygenase-2 (COX-2) inhibitor, in combination with a dendritic cell-based cancer vaccine significantly augments vaccine efficacy in reducing primary tumor burden, preventing metastasis, and increasing survival. This combination treatment was tested in MMTV-PyV MT mice that develop spontaneous mammary gland tumors with metastasis to the lungs and bone marrow. Improved vaccine potency was associated with an increase in tumor-specific CTLs. Enhanced CTL activity was attributed to a significant decrease in levels of tumor-associated IDO, a negative regulator of T cell activity. We present data suggesting that inhibiting COX-2 activity in vivo regulates IDO expression within the tumor microenvironment; this is further corroborated in the MDA-MB-231 human breast cancer cell line. Thus, a novel mechanism of COX-2-induced immunosuppression via regulation of IDO has emerged that may have implications in designing future cancer vaccines.     

X-linked inhibitor of apoptosis deficiency in the TRAMP mouse prostate cancer model

Deregulation of apoptotic pathways plays a central role in cancer pathogenesis. X-linked inhibitor of apoptosis protein (XIAP), is an antiapoptotic molecule, whose elevated expression has been observed in tumor specimens from patients with prostate carcinoma. Studies in human cancer cell culture models and xenograft tumor models have demonstrated that loss of XIAP sensitizes cancer cells to apoptotic stimuli and abrogates tumor growth. In view of these findings, XIAP represents an attractive antiapoptotic therapeutic target for prostate cancer. To examine the role of XIAP in an immunocompetent mouse cancer model, we have generated transgenic adenocarcinoma of the mouse prostate (TRAMP) mice that lack XIAP. We did not observe a protective effect of Xiap deficiency in TRAMP mice as measured by tumor onset and overall survival. In fact, there was an unexpected trend toward more aggressive disease in the Xiap-deficient mice. These findings suggest that alternative mechanisms of apoptosis resistance are playing a significant oncogenic role in the setting of Xiap deficiency. Our study has implications for XIAP-targeting therapies currently in development. Greater understanding of these mechanisms will aid in combating resistance to XIAP-targeting treatment, in addition to optimizing selection of patients who are most likely to respond to such treatment.     

Low-dose methotrexate enhances cycling of highly anaplastic cancer cells

We previously showed that cellular RedOx state governs the G₁-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F). This suggests that the G₁-S transition of AH130 cells depends on a respiration-linked step of DNA synthesis related to folate metabolism. In the study reported here, we characterized the effects of methotrexate (MTX), an inhibitor of dihydofolate-reductase, on the G₁-S transition of hepatoma cells, in the absence or the presence of exogenously added F, dihydrofolate (FH₂) or tetrahydrofolate (FH₄). MTX, at 1 μM or higher concentrations, inhibited G₁-S transition. This inhibition was completely removed by exogenous folates. Surprisingly, 10 nM MTX stimulated G₁-S transition. The addition of F, but not FH₂ or FH₄, significantly increased this effect. Furthermore, 10 nM MTX removed the block of the G₁-S transition operated by antimycin A or pyruvate, an effect which was enhanced in the presence of F. Finally, the stimulatory effect of 10 nM MTX was inhibited in the presence of serine. Our findings indicated that, under certain conditions, MTX may stimulate, rather than inhibiting, the cycling of cancer cells exhibiting a stem cell-like phenotype, such as AH130 cells. This may impact the therapeutic use of MTX and of folates as supportive care.     

A mouse prostate cancer model induced by Hedgehog overexpression

Summary Hedgehog is a regulatory protein during embryonic development and its abnormal activation in adult tissues has been implicated in tumorigenesis within sites where epithelial–mesenchymal interactions take place. In the prostate, Hedgehog signaling activation was observed during advanced cancer progression and metastasis, but whether Hedgehog overexpression can initiate prostate tumorigenesis remains unknown. We introduced a Hedgehog-expressing vector by intra-prostate injection and electroporation to address the effects of Hedgehog overexpression. The manipulation caused lesions with characteristic prostatic intraepithelial neoplasia or even prostatic cancer (CaP) phenotypes within 30 days, with Hedgehog overexpression demonstrated by immunohistochemistry and Western blot detections. The tumorigenic phenotypes were confirmed by discontinuity of basal cell marker p63, mix-up of CK-8/CK-18 positive epithelial cells in the stoma as well as absence of α-SMA positive fibro-muscular sheath. Comparable Hedgehog overexpression was found in human CaP specimen. Thus, Hedgehog overexpression induced prostate tumorigenesis starting from the normal status. Furthermore, a mouse prostate cancer model induced by Hedgehog overexpression was established and may be used for testing novel therapeutical approaches targeting at Hedgehog signaling pathway.These authors have contributed equally to this work.     

Low-dose radiotherapy effects the progression of anti-tumor response

The history of low-dose radiotherapy (LDRT or LDR) as a treatment modality for malignant tumors dates back to the 1920s. Even with the minimal total dose administered during treatment, LDRT can result in long-lasting remission. Autocrine and paracrine signaling are widely recognized for fostering the growth and development of tumor cells. LDRT exerts systemic anti-tumor effects through various mechanisms, such as enhancing the activity of immune cells and cytokines, shifting the immune response towards an anti-tumor phenotype, influencing gene expression, and blocking crucial immunosuppressive pathways. Additionally, LDRT has been demonstrated to enhance the infiltration of activated T cells and initiate a series of inflammatory processes while modulating the tumor microenvironment. In this context, the objective of receiving radiation is not to directly kill tumor cells but to reprogram the immune system. Enhancing anti-tumor immunity may be a critical mechanism by which LDRT plays a role in cancer suppression. Therefore, this review primarily focuses on the clinical and preclinical efficacy of LDRT in combination with other anti-cancer strategies, such as the interaction between LDRT and the tumor microenvironment, and the remodeling of the immune system.     

A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells

DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185^neu expressing tumor MBT-2 in mice. We found that recombinant LZ-8 stimulated mouse bone marrow-derived dendritic cells (DCs) via TLR4 and its stimulatory effect was not due to any microbe contaminant. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, LZ-8 cotreatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase in antitumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.     

CpG-enriched plasmid enhances the efficacy of the traditional foot-and-mouth disease killed vaccine

A CpG-enriched recombinant plasmid (pUC18-CpG) as an adjuvant of FMD killed vaccine was tested for immunization and vaccination challenge in a porcine model. Our preliminary results had indicated that the recombinant plasmid could enhance the humoral immune response triggered by the traditional oil-adjuvant vaccine after the initial inoculation. A subsequent vaccination-challenge test showed an increased PD(50) value. Thus, coadministration of the recombinant plasmid with the oil-adjuvant vaccine helped illicit an immune response earlier than that elicited by giving the vaccine alone. Our results showed that pUC18-CpG can be a potent immunoadjuvant for the traditional FMD killed vaccine and can greatly enhance the traditional vaccine's efficacy when given in combination with it.     

Immunogenicity and efficacy of an anthrax/plague DNA fusion vaccine in a mouse model

The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y .?pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B.?anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y.?pestis.     

Synergistic anti-tumor activity of isochaihulactone and paclitaxel on human lung cancer cells

Drug resistance frequently develops in tumors during chemotherapy. Therefore, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Here, we show that isochaihulactone (K8) enhanced paclitaxel-induced apoptotic death in human lung cancer cells, and the enhancing effect was related to increased NSAID-activated gene-1 (NAG-1) expression. CalcuSyn software was used to evaluate the synergistic interaction of K8 and paclitaxel on human lung cancer cells; the synergistic effect of K8 in combination with paclitaxel was increased more than either of these drugs alone. Furthermore, the activity of ERK1/2 was enhanced by the combination of K8 and paclitaxel, and an ERK1/2 inhibitor dramatically inhibited NAG-1 expression in human lung cancer cells. Therefore, this synergistic apoptotic effect in human lung cancer cells may be directly associated with K8-induced NAG-1 expression through ERK1/2 activation. Moreover, over-expression of NAG-1 enhanced K8/paclitaxel-induced apoptosis in human lung cancer cells. In addition, treatment of nude mice with K8 combined with paclitaxel induced phospho-ERK1/2 and NAG-1 expression in vivo. Targeting of NAG-1 signaling could enhance therapeutic efficacy in lung cancer. Our results reveal that activation of NAG-1 by K8 enhanced the therapeutic efficacy of paclitaxel in human lung cancer cells via the ERK1/2 signaling pathway.     

Characterization of the nuclear matrix proteins in a transgenic mouse model for prostate cancer

The nuclear matrix (NM) contains a number of proteins that have been found to be associated with transformation. We have previously identified changes in the NM associated with prostate cancer. In this study, we examine the molecular changes that are associated with prostate cancer development in transgenic adenocarcinoma of mouse prostate (TRAMP) model by studying the differences in the NM proteins (NMPs). We collected prostates from the TRAMP males at six critical time points: 6 weeks (puberty), 11 and 19 weeks (development of mild hyperplasia), 25 weeks (development of severe hyperplasia), 31 and 37 weeks (development of neoplasia). The nuclear matrices from the prostates collected at these time points were then isolated and the NMPs were characterized by high-resolution two-dimensional gel electrophoresis. We found three NMPs (E1A, E1B, and E1C) that were present in the 6-week-old prostate and two NMPs (E2A and E2B) that were present in the 11-week-old prostate. These NMPs were absent in the 31- and 37-week-old prostate. We also found five NMPs (E3A-E3E) that were present in the 31-week-old prostate, but absent in the earlier time points. In addition, three NMPs (Le1, Le2, Le3) were present at higher expression in the 6-, 11-, 19-, and 25-weeks old TRAMP prostates, but they were expressed lower during the development of neoplasia at 31- and 37-weeks old. Identification of these NMPs permits the development of novel markers that can characterize various stages of prostate cancer development as well as potentially therapeutic targets.     

Full length antigen priming enhances the CTL epitope-based DNA vaccine efficacy

Although CD8⁺ cytotoxic T lymphocyte (CTL) epitope-based DNA vaccination is valuable experience on vaccine research but many attempts are still continued to achieve acceptable protective response. To study the role of full length antigen in CTL epitope immunization, we evaluated cellular immunity of diverse patterns of complete Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) and the immunodominant CTL epitope (498–505) DNA injection in C57BL/6 mice. Optimal immune response was observed in the group immunized with the full length of gB in the first injection and CTL epitope in the second and third vaccination as assessed by lymphocyte proliferation assay (MTT), cytokine assay (ELISA) and CTL assay. B cell and spatially CD4⁺ T cell epitopes in full length protein might be important for appropriate priming of CTL immune response. These findings may have important implication for the improvement of CTL epitope based DNA vaccine against HSV and other pathogens.     

Rapamycin enhances docetaxel-induced cytotoxicity in a androgen-independent prostate cancer xenograft model by survivin downregulation

BackgroundDocetaxel is a first-line treatment choice in castration-resistant prostate cancer (CRPC). However, the management of CRPC remains an important challenge in oncology. There have been many reports on the effects of rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR), in the treatment of carcinogenesis. We assessed the cytotoxic effects of the combination treatment of docetaxel and rapamycin in prostate cancer cells. Furthermore, we examined the relationship between these treatments and survivin, which is a member of the inhibitory apoptosis family.MethodsProstate cancer cells were cultured and treated with docetaxel and rapamycin. The effects on proliferation were evaluated with the MTS assay. In addition, we evaluated the effect on proliferation of the combination treatment induced knockdown of survivin expression by small interfering RNA transfection and docetaxel. Protein expression levels were assayed using western blotting. PC3 cells and xenograft growth in nude mice were used to evaluate the in vivo efficacy of docetaxel and its combination with rapamycin.ResultsIn vitro and in vivo, the combination of rapamycin with docetaxel resulted in a greater inhibition of proliferation than treatment with rapamycin or docetaxel alone. In addition, in vitro and in vivo, rapamycin decreased basal surviving levels, and cotreatment with docetaxel further decreased these levels. Transfection siRNA against survivin enhanced the cytotoxicity of docetaxel in PC3 cells.ConclusionThe rapamycin-dependent enhancement of the cytotoxic effects of docetaxel was associated with the downregulation of survivin expression. Our results suggest that the combination of docetaxel and rapamycin is a candidate for the improved treatment of advanced prostate cancer.